WO2021211923A1 - Compositions and methods for treating disease - Google Patents

Abstract

The present disclosure relates to pharmaceutical compositions for the treatment or prevention of disease, including viral infections. Also disclosed are associated aerosol forms of such compositions, devices for producing and administering such aerosolized compositions, and associated methods of treating disease, including viral infections, and particularly infections resulting from coronavirus SARS-CoV-2.

Description

COMPOSITIONS AND METHODS FOR TREATING DISEASE

CROSS REFERENCE TO REUATED APPUICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Number 63/011,210, filed April 16, 2020, and U.S. Provisional Patent Application Number 63/033,450, filed June 2, 2020 the entire contents of which are incorporated herein by reference.

BACKGROUND

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus strain that causes coronavirus disease 2019 (COVID-19), a respiratory illness. As of April 16, 2020, there have been 2,081,969 total lab confirmed cases of SARS-CoV-2 infection in an ongoing pandemic. Beyond supportive care, there are currently no proven treatment options or US Food and Drug Administration (FDA)-approved drugs specifically for the treatment of SARS-CoV-2 and COVID-19 (CDC, 2020). Accordingly, there is an urgent need for therapeutic compositions and treatments for COVID-19, related SARS viral infections, and respiratory infections of all types.

Hydroxychloroquine is a 4-aminoquinoline compound that has been employed for decades for treating autoimmune conditions such as rheumatoid arthritis and lupus erythematosus, due to its immunomodulatory effects (Martindale: The Complete Drug Reference, 2021). It has been most frequently administered as hydroxychloroquine sulfate (HCQS) because this salt form is freely soluble in water (aqueous solubility of 1 in 5), with 1 mg of HCQS being equivalent to about 0.775 mg of the base (Martindale: The Complete Drug Reference, 2021). Absorption from the gastrointestinal tract is rapid and extensive (Tett et al., Br. J. clin. Pharmac. 1989, 27, 771-779). It then undergoes hepatic first pass metabolism and results in an oral bioavailability of 79% (Kaur et al., Journal of Anaesthesiology Clinical Pharmacology 2020, 36, S160-S165; Klimke et al., Medical Hypotheses 2020, 142, 109783).

The use of hydroxychloroquine to prevent and treat COVID-19 is based on its antiviral and immunomodulatory effects reported in the literature. Hydroxychloroquine showed in vitro antiviral activity against SARS-CoV-2 before and after infection in Vero cells, which were derived from kidney epithelial cells isolated from an African green monkey (Wang, M., et al, Cell Research 2020, 30, 269-271; Yao, X., et al, Clinical Infectious Diseases 2020, 71, 732-739). When the cells were pre-treated with the drugs for 2 hours before infection, the half maximal effective concentration (EC50) of hydroxychloroquine and chloroquine for inhibiting viral replication after 48 hours of incubation was 5.85 and 18.01 mM, respectively (Yao et al., 2020). From these in vitro data, it is evident that hydroxychloroquine is a promising drug for the prophylaxis and treatment of COVID-19. Although the antiviral mechanism of the drug is unclear, it has been shown to prevent the attachment of SARS-CoV-2 to angiotensin-converting enzyme 2 (ACE-2), sialic acid- containing glycoproteins, and gangliosides on the surface of host cells to which the vims needs to bind for entry (Fantini, J., et al, International Journal of Antimicrobial Agents 2020, 55, 105960; Pastick, K.A., et al, Open Forum Infectious Diseases 2020, 7, ofaal30). In addition, the drug is a weak base, so it increases the pH of the normally acidic endosomes and lysosomes in host cells. The alkalinity alters the homoeostasis of these intracellular organelles and hinders various processes in the viral life cycle that depend on them (e.g. cell entry, replication, release) (Fantini et al., 2020; Pastick et al., 2020).

The initial local airway inflammation in COVID-19 may lead to hypercytokinaemia, or “cytokine storm”, the uncontrolled upregulation of multiple pro-inflammatory cytokines such as interleukin (IE)-Ib, IF-6, IF-8, tumour necrosis factor-a, and granulocyte-colony stimulating factor (Romagnoli et al., Physiological Reviews 2020, 100, 1455-1466; Sun et al., Cytokine and Growth Factor Reviews 2020, 53, 38-42). The resultant hyperinflammation may then cause pulmonary fibrosis, hypoxaemia, damage to other organs, and death. Hydroxychloroquine has long been known to inhibit the production of some of the pro- inflammatory cytokines (Jang et al., Rheumatology 2006, 45, 703-710), and it is anticipated that early administration to COVID-19 patients will prevent the induction of a cytokine storm and further deterioration of health. In fact, early treatment of COVID-19 patients with orally administered hydroxychloroquine within one day of hospitalization (400 mg twice a day on Day 1, followed by 200 mg twice a day on Days 2 to 5) decreased their risk of being transferred to intensive care units by 53%, attributed to the anti-inflammatory properties of the drug (Fammers et al., International Journal of Infectious Diseases 2020, 101, 283-289).

Nonetheless, in vivo evidence supporting its clinical application is still lacking in many ways. Delivering this drug orally is inefficient when the lungs are the primary delivery target site. To achieve a therapeutic drug concentration in the airways, the oral dose needs to be sufficiently high to compensate for the drug loss due to first pass metabolism and distribution into other organs. However, high doses will increase the risk of systemic adverse effects. Indeed, oral hydroxychloroquine has been reported to cause cardiac toxicity including QT prolongation and ventricular arrhythmias in both COVID-19 patients and patients with other illnesses (rheumatoid arthritis, lupus erythematosus, malaria). This risk may be further heightened when when co-administered with azithromycin (Kaur et al., 2020; Pastick et ah, 2020). The lack of robustness of the clinical studies further complicates data interpretation. A number of them were not properly controlled, randomized, or blinded (Pastick et al., 2020). The patient sample size of some trials was too small for statistical power, while the disease severity amongst certain subject groups varied widely so it was difficult to interpret the results. Some published studies were not peer-reviewed, especially those from early 2020 as urgent dissemination of medical information on COVID-19 was prioritized (Pastick et al., 2020). The emergency nature of the pandemic may have imposed limitations on the design and execution of the studies, consequently impacting data quality. Because of its anti inflammatory properties, inhalation delivery of hydroxychloroquine solutions was tested in sheep for the treatment of asthma (Charous et al. 2001). Later research with a soft mist inhaler using an aqueous formulation of hydroxychloroquine sulfate proceeded into humans (Dayton et al., Respiratory Drug Delivery 2006, Davis Healthcare International Publishing, River Grove, IL, USA, pp. 429-432). The antiviral and anti-inflammatory activities of this drug against the rhinoviral infection in human bronchial cells were reported (Finkbeiner et al., Journal of Allergy and Clinical Immunology, 2004, 113, S264). Robust, controlled clinical trials for hydroxychloroquine utilizing a more efficient route of administration are required.

SUMMARY

Since the respiratory tract is the initial site of infection and inflammation, direct inhalation delivery is better targeted than oral administration, as lower doses can be used to achieve high local drug concentrations in the airways to maximize therapeutic action and minimize systemic adverse effects

Accordingly, provided herein are pharmaceutical compositions for the treatment of viral infections. In certain embodiments, the compositions are aerosols and treatment is effected upon inhalation and/or exhalation of the aerosol. Devices for producing the aerosol and/or administering the aerosol to a subject in need thereof are provided.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a schematic of a typical pressurized metered-dose inhaler.

FIG. 2 shows the relationship between aerodynamic diameter and lung deposition.

FIG. 3 shows a breath-actuated inhaler. FIG. 4 shows representative dose design options for dry powder inhalers.

FIG. 5 shows a soft mist inhaler.

FIG. 6 shows a representative mesh nebulizer inhaler.

FIG. 7 shows a representative jet nebulizer inhaler.

FIG. 8 shows absolute and relative doses of HCQS on the various parts of the dose output setup. The four bars represent 1 mL of 20 mg/mL (black), 1 mL of 50 mg/mL (white), 1 mL of 100 mg/mL (grey), and 1.5 mL of 100 mg/mL (hatch). Data presented as mean ± standard deviation (n = 9). Statistical difference indicated by * (p < 0.05).

FIG. 9 shows the volumetric median diameter of the droplets measured by laser diffraction. Data presented as mean ± standard deviation (n = 9). Statistical difference indicated by * (p < 0.05).

FIG. 10 shows the geometric standard deviation of the droplets measured by laser diffraction. Data presented as mean ± standard deviation (n = 9). Statistical difference indicated by * (p < 0.05).

FIG. 11 shows the percentage of aerosol by volume under 1, 2, 3, 5, and 10 pm measured by laser diffraction. Data presented as mean ± standard deviation (n = 9). Statistical difference indicated by * (p < 0.05).

FIG. 12 shows absolute (FIG. 12A) and relative doses (FIG. 12B) of HCQS on the various parts of the NGI setup. The four bars represent 1 mL of 20 mg/mL (black), 1 mL of 50 mg/mL (white), 1 mL of 100 mg/mL (grey), and 1.5 mL of 100 mg/mL (hatch). Data presented as mean ± standard deviation (n = 3).

FIG. 13 shows HCQ concentrations in whole blood (FIG. 13A), lung parenchyma (FIG. 13B), and heart (FIG. 13C) following intratracheal (GG), intravenous (IV), and oral (PO) administration to human subjects.

FIG. 14 shows pharmacokinetics in whole blood following administration of aerosolized HCQ as compared to IV and PO administration.

FIG. 15 shows plasma concentration of HCQ in Sprague Dawley rats following a single IT (FIG. 15A), IV (FIG. 15B), and PO (FIG. 15C) administration of HCQ sulfate. LLOQ = 1 ng/ml.

FIG. 16 shows blood concentration of HCQ in Sprague Dawley rats following a single IT (FIG. 16A), IV (FIG. 16B), and PO (FIG. 16C) administration of HCQ sulfate. LLOQ = 1 ng/ml.

FIG. 17 shows tissue concentration of HCQ in Sprague Dawley rats following a single GG (FIG. 17A), IV (FIG. 17B), and PO (FIG. 17C) administration of HCQ sulfate. DETAILED DESCRIPTION

Provided herein are pharmaceutical compositions for the treatment or prevention of disease, such as viral infections, and associated aerosol forms of such compositions, devices for producing and administering such aerosolized compositions, and associated methods of treating disease, such as viral infections. Aerosol formulations delivered via inhalation advantageously result in higher drug concentration in the airway and lung as compared to blood and plasma, promoting greater local efficacy, lower systemic exposure, and greater safety.

Pharmaceutical Compositions

In one aspect, provided herein is a pharmaceutical composition for inhalation, comprising a compound selected from the group consisting of hydroxychloroquine, primaquine, mepacrine, mefloquine, pamaquine, pentaquine, tafenoquine, lumefantrine, pyronaridine, and salts thereof; and a carrier.

In certain embodiments, the composition further comprises one or more of: a tonicity agent, a flavoring agent, a preservative, a surfactant, a stabilizer, a pH adjustment agent, and a propellant.

In certain embodiments, the composition is formulated ( e.g ., formulated to be delivered) for inhalation by mouth or nose. In certain embodiments, the composition is formulated (e.g., formulated to be delivered) for exhalation by mouth or nose.

In certain embodiments, the compound is hydroxychloroquine (HCQ), or a pharmaceutically acceptable salt thereof. In certain embodiments, the compound is (R)- hydroxychloroquine, or pharmaceutically acceptable salt thereof. In certain embodiments, the compound is (S)-hydroxychloroquine, or pharmaceutically acceptable salt thereof. In certain embodiments, the compound is a sulfate salt. In certain embodiments, the compound is a monosulfate salt. In certain embodiments, the compound is a mixed salt. In certain embodiments, it is a mixture of the R and S forms HCQ, which may be in the form of various salts.

In certain embodiments, the composition is a dry powder. In certain embodiments, the weight percent of compound in the composition is at least 80%, at least 90%, at least 95%, or 100%. In certain embodiments, the dry powder is formulated for direct inhalation. In certain embodiments, the dry powder is formulated for reconstitution as a solution. The concentration of the HCQ may be varied to accommodate other substances to adjust the solution properties including osmolarity, pH, irritation and flavor as needed. In certain embodiments, the concentration of the compound in the composition is in the range of 0.01- 1M. In certain embodiments, the concentration of the compound in the composition is in the range of 5mg/ml to 100 mg/ml. In certain embodiments, the concentration of the compound in the composition is in the range of 5mg/ml to 200 mg/ml. In a particular embodiment, the concentration of the compound (e.g., hydroxychloroquine sulfate) is 20 mg/ml. In another particular embodiment, the concentration of the compound (e.g., hydroxychloroquine sulfate) is 50 mg/ml. In another particular embodiment, the concentration of the compound (e.g., hydroxychloroquine sulfate) is 100 mg/ml. In certain embodiments, higher concentrations may be achieved. For example, higher concentrations of hydroxychloroquine, or a salt thereof, may be obtained up to the limit of solubility of those salts. Adjustment of pH may be used to achieve higher solubility is also possible. Solubilization using co-solvents, surfactants, or liposomes, formulation as a suspension, and other methods known in the art may also be used to achieve increased concentrations of solution.

In certain embodiments, the carrier is a liquid carrier. In certain embodiments, the liquid carrier comprises water, saline, ethanol, or a combination thereof. In certain embodiments, the water is deionized, microfiltered, and/or sterilized water.

In certain embodiments, the composition is a solution, microemulsion, suspension, or co-suspension.

In certain embodiments, the compound is present in the form of a microparticle, nanoparticle, cyclodextrin complex, liposome, or noisome.

In certain embodiments, the composition is formulated for aerosolization.

In certain embodiments, the composition is an aerosol. The aerosol may be produced by a device as described herein. In certain embodiments, the device is a nebulizer. In a particular embodiment, the nebulizer is a vibrating mesh nebulizer. The aerosol comprises droplets, each having a median volumetric diameter in the range of 2-8 pm. In certain embodiments, each droplet has a median volumetric diameter in the range of 4-6 pm. In a particular embodiment, each droplet has a median volumetric diameter in the range of 4.3-5.2 pm, for example, with about 50-60% of the aerosol by volume having droplets with a median volumetric diameter of less than 5 pm. In another particular embodiment, each droplet has a median volumetric diameter in the range of 4.3-5.0 pm (e.g., 4.34-4.95).

In certain embodiments, the composition is formulated to suppress flavor and/or irritation. In certain embodiments, the composition comprises a flavoring agent. In certain embodiments, the flavoring agent comprises a GRAS (generally recognized as safe) material. In certain embodiments, the flavoring agent is a plant extract, such as a terpene. In a particular embodiment, the terpene is menthol. In certain embodiments, the flavoring agent is a topical anesthetic, e.g., benzocaine. In certain embodiments, the concentration of the flavoring agent is in the range of 0.001M-1M. In certain embodiments, the concentration of the flavoring agent is in the range of O.Olmg/g-lOO mg/g. An agent useful for flavor and/or irritation suppression may be used in the form of a solid, gel, solution, microemulsion, emulsion, liposome, microparticle (e.g., smaller than 5 pm), nanoparticle, clathrate.

In certain embodiments, the composition comprises a preservative, wherein the preservative inhibits decomposition of the compound, or prevents bacterial growth in the composition. In certain embodiments, the preservative is an antioxidant. In certain embodiments, the antioxidant is a non-ionic organic molecule, or an inorganic or organic ionic molecule, or a salt thereof. In certain embodiments, the preservative is a hydroxylated organic molecule (e.g., citrate, phenol, or a phenol derivative, e.g., hydroxyl methoxybenzoate) .

In certain embodiments, the composition comprises a surfactant. In certain embodiments, the surfactant is an ionic or non-ionic surfactant. In certain embodiments, the composition comprises a stabilizer. In certain embodiments, the surfactant is an ionic or non ionic stabilizer.

In certain embodiments, the composition comprises a propellant. In certain embodiments, the propellant is a gas, or a halogenated or nonhalogenated hydrodrocarbon. In certain embodiments, the propellant is carbon dioxide, propane, butane, or isobutane. In certain embodiments, the propellant is a chlorofluorocarbon, or a fluorocarbon (e.g., a hydrofluoroalkane) .

In certain embodiments, the composition comprises a pH adjustment agent. In certain embodiments, the pH adjustment agent is an acid or a base (e.g., hydrochloric acid or sodium hydroxide). In certain embodiments, the pH of the composition is in the range of 6.0 to 7.8. In certain embodiments, the pH of the composition is in the range of 7.0 to 7.4.

In certain embodiments, the composition comprises a tonicity agent. In certain embodiments, the composition is hypotonic. In certain embodiments, the composition is isotonic or isosmolar. In certain embodiments, the composition is hypertonic.

In certain embodiments, the viscosity of the composition is in the range of 0.8- 1.6

Pa-s.

In certain embodiments, the osmolality of the composition is in the range of 100-1000 mmol/kg. Preferably, the composition is sterile. In certain embodiments, sterlization is achieved by sterile filtration into a sterile container. In certain embodiments, thermal sterilization may be used. In certain embodiments, sterilization by exposure to radiation may be used.

In certain embodiments, the composition further comprises one or more additional therapeutic agents. In certain embodiments, the one or more additional therapeutic agents are independently an anti-viral, an anti-coagulant, an anti-parasitic, an anti-microbial, an anti fungal, an anesthetic, a bronchodilator, a steroid, an anti-inflammatory, or a vitamin. In a particular embodiment, the additional therapeutic agent comprises zinc, for example, a salt or complex comprising zinc (II) cations. Representative zinc salts include zinc (II) acetate, zinc (II) sulfate, zinc (II) chloride, zinc (II) gluconate, zinc (II) picolinate, zinc (II), orotate, or zinc (II) citrate. The additional therapeutic agents may be pre-mixed into a single formulation. Alternatively, the additional therapeutic agent may be provided in the form of a separate formulation that is co-administered with the formulation comprising the compound ( e.g ., hydroxychloroquine). In certain embodiments, for example, the additional therapeutic agent is provided in a separate part of a device (e.g., a separate vial for nebulization, or a separate compartment in a dry powder inhaler), such that the compound (e.g., hydroxychloroquine) and the additional therapeutic are co-administered to a patient by the device during the same inhalation. Such co-administration provides convenience to patients, delivers the components to the same site for additive or synergistic activity, and improves greater compliance with the medications due to easier use.

In certain embodiments, the composition is stable at 25 °C for at least 15 days, at least 30 days, or at least one year. In certain embodiments, the composition is stable at 40 °C for at least 15 days, at least 30 days, or at least one year. In a particular embodiment, the composition is stable for at least 15 days in the dark. In another particular embodiment, the composition is stable for at least 30 days in the dark. In another particular embodiment, the composition is stable for at least one year in the dark.

In certain embodiments, the subject is human. In certain embodiments, the subject is a non-human animal.

Methods of Delivery

SARS-CoV-2 is found through the respiratory tract, including the mouth, nose, large airways, bronchioli and alveoli of subjects having COVID-19. Depending on the sites of the infection, different methods of delivery and formulation may be judiciously applied either using the same formulation and device, or combinations of them. Aerosolized HCQ can be inhaled via the mouth and exhaled via the nose to cover much of the whole of respiratory tract. Localized delivery to the nose may be achieved with many different devices known to those familiar with the art, such as nasal sprays, insufflators, or nasal rinses. Delivery to selected parts of the respiratory tree may be achieved with bronchoscopic sprays. The combination of particle size, inspiratory flow rate and placing the aerosol at different portion of the inspiration has been also used for targeting aerosols to specific regions of the respiratory tract (WD Bennett, “Targeting Respiratory Drug Delivery with Aerosol Boluses,” Journal of Aerosol Medicine Vol. 4, No. 2).

The methods of delivery preferably are such as to minimize to possibility of exposure of the caregivers to the viral infection by the subject with COVID-19. In some embodiment, the device generating the aerosol will be such that excess aerosol and the exhalation by the patients will go through a filter minimizing the possibility of the vims getting in the environment, Exhalation through the nose may be prevented by wearing nose clips whereas exposure via exhalation through the mouth may be minimized with a face mask. In some embodiments, the patient may be inhaling and exhaling in an enclosure such as helmet or hood.

Aerosols

In another aspect, provided herein is an aerosol comprising a pharmaceutical composition as described herein. Aerosols may comprise solid particles, semi-solid particles, liquid particles (i.e., droplets), or mixtures thereof. Compounds used in the form of solid or semi-solid particles may be encapsulated or complexed in order to achieve favorable or advantageous properties such as size, weight, solubility, and dispersibility. The size of aerosol particles can be controlled by a device used to produce such particles. Particle ( e.g ., droplet) size and distribution and deposition in the respiratory tract will result from the device used and the inhalation pattern of the subject. See, e.g., Laube BL, et al. Eur. Respir. J. 2011, 37, 1308-1311.

Devices

In another aspect, provided herein is a device for delivering to a subject via inhalation or exhalation (e.g., oral or nasal) an aerosol as described herein, i.e., an aerosolized form of a pharmaceutical composition as described herein. In certain embodiments, the device is a nebulizer (e.g., jet or ultrasonic), atomizer, vaporizer, or electrospray. In certain embodiments, the device is propellant-driven, breath-actuated, or pump-actuated. In certain embodiments, the device comprises a metering valve. In certain embodiments, the device is configured to control or regulate aerosol droplet or particle size. In certain embodiments, the device is configured to produce the aerosol for a duration in the range of 5 seconds to 30 minutes. In certain embodiments, the device is configured to control or regulate aerosol velocity. In certain embodiments, the device comprises a filter, wherein the filter is positioned to receive the subject’s exhalation or the excess aerosol from the device. In certain embodiments, the filter is a HEPA filter.

In certain embodiments, the device is configured to deliver an amount of the composition in the range of 1-130 mg, e.g., 1-5 mg, 5-10 mg, 10-20 mg, 10-50 mg, 25-75 mg, 50-75 mg, 75-100 mg, 75-130 mg, or 100-130 mg. In certain embodiments, a dose of compound in the range of 1-130 mg, e.g., 1-5 mg, 5-10 mg, 10-20 mg, 10-50 mg, 25-75 mg, 50-75 mg, 75-100 mg, 75-130 mg, or 100-130 mg. In certain embodiments, the device is configured to deliver the composition to the respiratory tract with an efficiency (i.e., percent compound delivered) of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%.

In certain embodiments, the device is configured to deliver an amount of the composition in the range of 1-300 mg, e.g., 1-5 mg, 5-10 mg, 10-20 mg, 10-50 mg, 25-75 mg, 50-75 mg, 75-100 mg, 75-150 mg, 100-150 mg, 150-200 mg, 200-250 mg, or 250-300 mg. In certain embodiments, a dose of compound in the range of 1-300 mg, e.g., 1-5 mg, 5-10 mg, 10-20 mg, 10-50 mg, 25-75 mg, 50-75 mg, 75-100 mg, 75-150 mg, 100-150 mg, 150-200 mg, 200-250 mg, or 250-300 mg. In certain embodiments, the device is configured to deliver the composition to the respiratory tract with an efficiency (i.e., percent compound delivered) of at least 15%, at least 25%, at least 50%, at least 60%, at least 70%, or at least 90%.

In certain embodiments, the device is a metered-dose inhaler. See, e.g., Newman SP. “Principles of metered-dose inhaler design.” Respir Care. 2005 Sep; 50(9): 1177-88; Roche N, Dekhuijezen R. “The evolution of pressurized metered-dose inhalers from early to modern devices.” J Aerosol Med Pulm Drug Deliv. 2016; 29(4):311.

In certain embodiments, the device comprises a non-pressurized reservoir containing the composition. In certain embodiments, the device a pressurized reservoir containing the composition. See, e.g., Cogan PS, Sucher BJ. “Appropriate use of pressurized metered-dose inhalers for asthma.” US Pharm. 2015; 40(7), 36-41; Vehring R, Ballesteros DL, Joshi V, Noga B, Dwivedi SK. “Co-suspensions of microcrystals and engineered microparticles for uniform and efficient delivery of respiratory therapeutics from pressurized metered dose inhalers.” Langmuir 201228(42), 15015-15023.

In certain embodiments, the device is a breath-actuated inhaler (FIG. 3). In certain embodiments, the device is dry powder inhaler (FIG. 4). See, e.g., Islam N, Gladki E. “Dry powder inhalers (DPIs)-a review of device reliability and innovation.” Int J Pharm. 2008; Daniher DI, Zhu J. “Dry powder platform for pulmonary drug delivery.” Particulogy. 2008 Aug. Dry powder inhalers may be breath-actuated, may deliver particles having a mean mass aerodynamic diameter (MMAD) of less than 5 microns, and may produce inspiratory flow rates of 30-60 L/min. Other soft mist inhalers such as AERx and Medspray have been in development based on extrusion of liquids through arrays of nozzles (Dayton et al. 2006).

In certain embodiments, the device is a soft mist inhaler (FIG. 5). See, e.g., Dalby RN, Eicher J, Zierenberg B. “Development of Respimat Soft Mist inhaler and its clinical utility in respiratory disorder.” Med Devices. 2011 Aug. In certain embodiments, the device is a Respimat® Soft Mist inhaler.

In certain embodiments, the device is an Aerogen® Ultra/ Aerogen® Solo nebulizer (FIG. 6). The device is manufactured by Aerogen Ltd. Aerogen® Ultra is an accessory specific to the Aerogen® Solo nebulizer. This device facilitates intermittent and continuous nebulization and optional supply of supplemental oxygen to pediatric (29 days or older) and adult patients in hospital use environments via mouthpiece or aerosol face mask. The Aerogen® Ultra is a single patient use device. In certain embodiments, the device is used intermittently for a maximum of 20 treatments, which is based upon a typical usage profile of four 3 ml doses per day over 5 days, with an average treatment time of 9 minutes. In other embodiments, the device is used continuously for a maximum of 3 hours. In other embodiments the device uses a volume of approximately 0.5 mL that can be delivered in about 1 minute.

In certain embodiments, the device is a PARI LC Sprint (FIG. 7), used in conjunction with a compressor. The PARI LC Sprint is manufactured by PARI Respiratory Equipment, Inc.

In certain embodiments, the device is a soft mist inhaler manufactured by Medspray (Enschede, Twente, Netherlands). Such devices include the following: ADI/Colistair (puff size 50 pL, capacity 1 mL); PFSI (puff size 30 pL, capacity 90 pL); Ecomyst90 (puff size 25 pL, capacity 5 or 10 mL); and Pulmospray, Pulmospray ICU devices (patient breaths in through mouth and out through nose).

The present disclosure contemplates specific combinations of the compositions and devices disclosed herein. Therapeutic methods

In another aspect, provided herein is a method of treating or preventing inflammation associated with a viral infection in a subject, comprising administering to the subject via inhalation an aerosol as described herein, i.e., an aerosolized pharmaceutical composition as described herein.

In another aspect, provided herein is a method of treating or preventing a viral infection in a subject, comprising administering to the subject via inhalation an aerosol as described herein, i.e., an aerosolized pharmaceutical composition as described herein.

In certain embodiments, the administration further comprises exhalation of the aerosol. In certain embodiments, the viral infection is an infection of a rhinovirus ( e.g ., human rhinovirus) or a coronavirus in the subject. In certain embodiments, the coronavirus is SARS-CoV-2.

In certain embodiments, the method comprises administering an amount of aerosol containing a dose of the compound in the range of 1-130 mg. In certain embodiments, the dose of the compound (e.g., hydroxychloroquine sulfate) is in the range of 5-100 mg. In certain embodiments, the dose of the compound (e.g., hydroxychloroquine sulfate) is in the range of 5-90 mg. In certain embodiments, the dose of the compound (e.g., hydroxychloroquine sulfate) is in the range of 5-80 mg. In certain embodiments, the dose of the compound (e.g., hydroxychloroquine sulfate) is in the range of 5-70 mg. In certain embodiments, the dose of the compound (e.g., hydroxychloroquine sulfate) is in the range of 5-60 mg. In certain embodiments, the dose of the compound (e.g., hydroxychloroquine sulfate) is in the range of 5-50 mg. In certain embodiments, the dose of the compound (e.g., hydroxychloroquine sulfate) is in the range of 10-100 mg. In certain embodiments, the dose of the compound (e.g., hydroxychloroquine sulfate) is in the range of 10-90 mg. In certain embodiments, the dose of the compound (e.g., hydroxychloroquine sulfate) is in the range of 10-80 mg. In certain embodiments, the dose of the compound (e.g., hydroxychloroquine sulfate) is in the range of 10-70 mg. In certain embodiments, the dose of the compound (e.g., hydroxychloroquine sulfate) is in the range of 10-60 mg. In certain embodiments, the dose of the compound (e.g., hydroxychloroquine sulfate) is in the range of 10-50 mg.

In certain embodiments, the method comprises administering the aerosol for a duration of time in the range of 1 second to 1 hour. In particular embodiments, the duration of time may be in the range of 1 second to 30 minutes, 10 seconds to 30 minutes, 30 seconds to 30 minutes, 1-30 minutes, 2-30 minutes, 3-30 minutes, 4-30 minutes, 5-30 minutes, 6-30 minutes, 7-30 minutes, 8-30 minutes, 9-30 minutes, 10-30 minutes, 15-30 minutes, 20-30 minutes, or 25-30 minutes. In other particular embodiments, the duration of time may be in the range of 1 second to 20 minutes, 10 seconds to 20 minutes, 30 seconds to 20 minutes, 1- 20 minutes, 2-20 minutes, 3-20 minutes, 4-20 minutes, 5-20 minutes, 6-20 minutes, 7-20 minutes, 8-20 minutes, 9-20 minutes, 10-20 minutes, or 15-20 minutes. In other particular embodiments, the duration of time may be in the range of 1 second to 10 minutes, 10 seconds to 10 minutes, 30 seconds to 10 minutes, 1-10 minutes, 2-10 minutes, 3-10 minutes, 4-10 minutes, 5-10 minutes, 6-10 minutes, 7-10 minutes, 8-10 minutes, or 9-10 minutes, 10-30 minutes, 15-30 minutes, 20-30 minutes, or 25-30 minutes.

In certain embodiments, the method comprises administering the aerosol to the subject one time per day, up to a maximum daily compound dosage of 800 mg. In certain embodiments, the method comprises administering the aerosol to the subject 2 or more times per day, up to a maximum daily compound dosage of 800 mg. In certain embodiments, the administration comprises a single inhalation of the maximum amount of aerosol that is tolerated by the subject, up to the maximum daily dose of 800 mg. In certain embodiments, the administration comprises multiple inhalations of the maximum amount of aerosol that is tolerated by the subject, up to the maximum daily dose of 800 mg. In certain embodiments, the number of inhalations per day is 1-8, 1-4, or 4.

In certain embodiments, the method comprises administering the aerosol to the subject via a ventilator. For example, a nebulizer may be connected to a ventilator to which a subject is connected.

In certain embodiments, treating the viral infection comprises reducing viral count and/or relieving clinical symptoms such as fever, chills, and pain. In certain embodiments, the viral load of the subject is reduced by at least 50% ( e.g ., 50-90%) within 24 hours, at least 50% (e.g., 50-90%) within 2 days, at least 50% (e.g., 50-90%) within 3 days, at least 50% (e.g., 50-90%) within 4 days, at least 50% (e.g., 50-90%) within 5 days, at least 50% (e.g., 50-90%) within 6 days, at least 50% (e.g., 50-90%) within 7 days, at least 50% (e.g., 50-90%) within 8 days, at least 50% (e.g., 50-90%) within 9 days, or at least 50% (e.g., 50-90%) within 10 days. In some embodiments, the viral load is reduced by 90-100% within 2-10 days.

In certain embodiments, the aerosol is delivered to the lungs of the subject with an efficiency in the range of 10-70%, e.g., about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%.

In certain embodiments, the method further comprises administering an agent which masks the flavor of, or other physical sensations associated with, hydroxychloroquine, or a salt thereof. Administration of such an agent may be prior to, concurrently with, or following administration of the hydroxychloroquine, or salt thereof. Suitable flavor masking agents include those described herein, e.g., terpenes such as menthol, topical anesthetics such as benzocaine, or a combination thereof.

In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject has a pre-existing condition or comorbidity, e.g., the subject has or has a history of smoking, obstructive pulmonary/airway disorder, or immunodeficiency. In certain embodiments, the subject is receiving or has received treatment of a pre-existing condition or comorbidity. The present disclosure contemplates patient selection strategies, wherein subjects that respond particularly well to the disclosed methods are selected on the basis of characteristics such as age, BMI, lung capacity, VO 2 max, viral count, and disease and/or symptom severity.

In certain embodiments, treatment or prevention begins prior to the onset of symptoms in the subject, or as a means of prophylaxis of persons at risk from the infection. In certain embodiments, treatment begins concurrently with the onset of symptoms. In certain embodiments, treatment begins after the onset of symptoms (e.g., 1 hour, 12 hours, 24 hours,

2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, or 9 days after the onset of symptoms). SARS-CoV-2

Although the molecular mechanism of action of HCQ against SARS-CoV-2 and in the treatment of COVID-19 has not been fully elucidated, findings from previous studies have suggested that HCQ may inhibit the coronavirus through a series of steps. Firstly, the drugs can change the pH within the airway epithelial cells, inhibiting the fusion of the virus to the cell membrane. They may also inhibit nucleic acid replication, glycosylation of proteins, virus assembly, new virus particle transport, virus release and other processes to achieve antiviral effects. The anti-inflammatory properties of CQ/HCQ should also be considered since they may help mediate the cytokine storm that that has been suggested to play a role in clinical worsening after SARS-CoV-2 infection (Fox, 1993; Mehta et ak, 2020; Savarino, Boelaert, Cassone, Majori, & Cauda, 2003).

Rationale for using aerosol HCQ (AHCQ) for prevention and treatment of SARS- CoV-2 and COVID-19 include the following:

HCQ has potent antiviral activity against SARS-CoV-2 in vitro.

HCQ is known to have immunomodulatory effects, on top of the in vitro antiviral activity, and immune modulation is believed to play a role in the treatment of COVID-19.

SARS-CoV-2 infects individuals through the respiratory tract and COVID-19 manifests initially as a respiratory disease.

In silico data suggests that oral HCQ at very high doses achieves high lung tissue concentrations after administration for several days. AHCQ may achieve comparable or higher lung concentrations, and also high airway concentrations, earlier, and with a lower risk of systemic toxicity. This is of importance when considering the use of HCQ for the treatment of COVID-19, since many of the patients and at-risk populations are more prone to systemic toxicities of oral HCQ.

In a phase I study from 2004 an orally inhaled solution of HCQ was administered to participants at a concentration of 100 mg/ml, equivalent to 300,000 mM. The plasma PK showed a very short rapid spike followed by slow systemic elimination leading to low systemic concentrations. This suggests that the drug was highly bound to the lung tissue. The solution concentration vastly exceeds the in vitro EC50 for SARS-CoV-2. Even after rapid dilution by a couple magnitudes of order in the airway lumen due to the resident liquid and absorption, the concentration in the airways may be higher than the EC50. See, F. Dayton, S.G. Owen, D. Cipolla, A. Chu, B. Otulana, G. Di Sciullo, and B.L. Charous, “Development Of An Inhaled Hydroxychloroquine Sulfate Product Using The Aerx® System To Treat Asthma” Respiratory Drug Delivery, 2006, the contents of which is incorporated by reference.

Chronic use of AHCQ may achieve high lung and airway drug concentrations for a prolonged period of time, without a significant risk of systemic toxicity. This attribute may have therapeutic applications in prophylaxis for SARS-CoV-2.

In vitro EC50 values for inhibition of SARS-CoV-2 by HCQ decreased with longer incubation times, suggesting that incubation time may influence the drug’s antiviral activity. AHCQ may achieve prolonged exposure of lung tissue and airways to HCQ with a reduced risk for systemic toxicity.

It is also expected that inhaled HCQ will reach immediately therapeutic concentrations in the lung, in contrast to oral HCQ which is known to have very long time to equilibrate with a terminal half-life of elimination of about 1000 hours (Tett et ah, Br. J. Clin. Pharmac. 1988, 26, 303-313).

Method of Preparation

In another aspect, provided herein is a method of preparing a composition as described herein, comprising the step of selecting an acid addition salt of the compound, and optionally selecting one or more pH adjustment agents, such that the pH of the composition is in the desired pH range ( e.g ., in the range of 6.0-7.8, or in the range of 7-7.4).

Kits

In another aspect, provided herein is a kit for preparing a composition as described herein, comprising the composition components and instructions for preparing the composition.

Examples

Example 1. Estimates of the concentration and daily doses (mL loaded in the nebulizer) of aerosol HCQ to prevent and treat COVID19 respiratory infections.

The goal is to use the highest safe and tolerable dose and concentration that is likely to have adequate anti-COVID19 activity in the airways. The following assumptions are made:

1) The maximum dose at this point is limited by the lung dose found to be safe and tolerable in a Phase 2 asthma study: about 13-14 mg.

2) The maximum concentration is limited by CMC work and the safety and tolerability.

3) The minimum target concentration in the liquid in the airway lumen based on the in vitro activity of HCQ against COVID19 is about 100 mM (the activity measured in vitro is by sustaining this concentration in the supernatant where in humans the concentration will go down with time with a half-life of about 8 hours based on a previous Phase 1 PK study; also there was improved in vitro activity by sustaining that concentration over 2 days)

4) The dilution factor based on other studies with approved inhaled antibiotics and rhDNase using nebulizers and similar volumes of solution calculated as the formulation concentration in the nebulizer/C _max in sputum is about lOOOx.

The required concentration in the nebulizer to achieve a concentration of at least 100 mM in the airway lumen is expected to be about 0.034 mg/ml. Therefore the desired minimum concentration in the nebulizer is > 34 mg/mL (1000x0.034).

The required volume of the formulation in the nebulizer to achieve the desired dose is determined as follows. To achieve a lung dose of 14 mg (assuming that the efficiency of the nebulizer device is 30%), the volumes needed for various concentrations are:

It is noted that the actual volumes may need to be somewhat higher as the dead volume is about 100 microliters and some binding of HCQ to the nebulizer may occur.

Example 2. Formulations tested in the Aero gen Ultra and Pari LC Sprint nebulizers. 1) 100 mg/mL HCQ sulfate in water, adjusted to be isosmolal with isotonic saline.

2) Dilutions down to 20 mg/mL HCQ Sulfate of the final formulation above with isotonic saline. Solutions are sterilized by sterile filtration.

Example 3. Formulation and device

Hydroxychloroquine Sulfate (CAS registry number 747-36-4), “AHCQ”:

The quantitative composition of the drug product is summarized in Table 1 and Table 2.

Table 1. Quantitative Composition of 5 mg/mL AHCQ in 5 mL Vials

Table 2. Quantitative Composition of 100 mg/mL AHCQ in 5 mL Vials

The finished product is Hydroxychloroquine Sulfate, USP sterile solution in a 5-mL vial. Two concentrations will be prepared, 5 mg/mL and 25 mg/mL, to cover the range of solution concentrations necessary for product testing and for administration during Phase 1 studies.

The finished product will be a solution for inhalation delivered by a nebulizer, used either undiluted, or further diluted using 0.9% Sodium Chloride Injection, USP.

The manufacturing process is briefly described in the table below.

All excipients used in the manufacturing of AHCQ drug product will be USP/NF compendial grade. Dilution solutions will be 0.9% Sodium Chloride Injection, USP in 100 mL, 500 mL, or 1000 mL PVC containers with lot numbers and expiry dates. All products are stored according to label temperature requirements in a secure alarmed licensed drug storage room with continuous temperature and humidity monitoring.

The nebulizer to be used for AHCQ delivery is the Aerogen Solo Nebulizer (FIG. 6) with standard adult mouthpiece. As an alternative, the Aerogen Ultra may be used with the exhaust pipe with a filter. The Aerogen device can provide the patient with up to 9-fold higher drug dose than a standard small volume nebulizer (SVN) during mechanical ventilation. This device was cleared by FDA on October 17, 2014 via the 510(k) pathway (K133360). The Aerogen Solo utilizes active vibrating mesh technology, where energy applied to the vibrational element, causes vibration of each of the 1000 funnel shaped apertures within the mesh. The mesh acts as a micropump drawing liquid through the holes producing a low velocity aerosol optimized for targeted drug delivery to the lungs.

On inhalation, the air is drawn through the inlet valve on the base of the device creating a flow of air or oxygen through the device. This purges the aerosol chamber of aerosol and delivers drug to the patient via the mouthpiece. When the patient breaths out, the inlet valve closes and the exhalation valve on the mouthpiece opens. This allows the patient to exhale through the port on the mouthpiece while the aerosol chamber is refilled by the Aerogen Solo.

Example 4. Aerosol formulations and characterization Materials

Hydroxychloroquine sulfate (HCQS) powder of United States Pharmacopoeia (USP) grade (Lot 1910P031, Batch 033600-192021) was purchased from Sci Pharmtech Inc (Taoyuan, Taiwan); sodium chloride and orthophosphoric acid from Thermo Fisher Scientific (Waltham, MA, USA); and sodium hydroxide and sodium 1-pentanesulfonate monohydrate from Sigma- Aldrich (St Louis, MO, USA). Chromatographic grade methanol and acetonitrile were bought from RCI Labscan (Bangkok, Thailand) and Honeywell (Morris Plains, NJ, USA), respectively. Deionised water was obtained from a MODULAB^® High Flow Water Purification System (Evoqua Water Technologies, Pittsburgh, PA, USA).

Preparation of HCQS nebulised solutions

A 0.55% w/v solution of sodium chloride in deionised water was made as the diluent for the HCQS solutions. Ten grams of HCQS powder per 100 mL of the final solution volume was weighed into a glass volumetric flask. The HCQS was dissolved by adding the 0.55% w/v sodium chloride diluent to approximately 60% of the final volume. Twenty four millilitres of 0.1 N sodium hydroxide was then added to the volumetric flask per 100 mL of the final volume. Small amounts of white precipitate initially appeared upon adding the sodium hydroxide solution but they quickly dissolved within a second. The final volume was made up with the sodium chloride diluent to obtain 100 mg/mL of HCQS (Table 1). Solutions at 20 and 50 mg/mL were prepared by diluting the 100 mg/mL solution accordingly with normal saline (0.9% w/v sodium chloride in deionised water). All HCQS solutions were transferred from volumetric flasks to 50 mL polypropylene centrifuge tubes (Coming, Corning, NY, USA) and stored in darkness at ambient temperature until use. Measurement of osmolality and pH

The osmolality of the 20, 50, and 100 mg/mL HCQS solutions was measured with a K-7000 vapor pressure osmometer (Knauer, Berlin, Germany). The cell and head temperatures were set as 60°C and 62°C, respectively, and allowed to stabilise for an hour before use. These temperatures followed those recommended in the instrument manual for calibrating and measuring sodium chloride aqueous solutions (K-7000 Vapor Pressure Osmometer User Manual V7109, 2007). The measurement time and gain were 1.5 minutes and 16, respectively. Approximately 1 mL of each sample solution was drawn into glass microsyringes and inserted into the osmometer. One droplet from each sample was dispensed onto the thermistor for each osmolality measurement. The droplet was replaced by a new one when repeating the measurement. The experiments were conducted in quadruplicate (n = 4) for each HCQS solution. The target osmolality range was 260-360 mOsmol/kg H2O. The pH of the solutions was measured with a pH 700 benchtop meter (Oakton, Vernon Hills, IL, USA). The target pH range was 6.8-7.5.

High performance liquid chromatography (HPLC)

HCQS was quantified by a modified reverse phase-HPLC method from the USP (United States Pharmacopeia 43 - National Formulary 38, 2020). The assay was performed on an automated HPLC system consisted of DGU-20A degassing unit, a LC-20AT HPLC pump, a SIL-20A HT autosampler, a CTO-20A column oven and an SPD-20A UV detector (Shimadzu, Kyoto, Japan). The mobile phase was composed of 10:10:80:0.2 by volume of methanol, acetonitrile, 0.12 g/L sodium 1 -pentane sulfonate monohydrate aqueous solution, and orthophosphoric acid. The mobile phase and all other solvents were filtered and degassed before use. The Agilent Zorbax SB-C18 column (5 pm, 4.6 x 250 mm; Waters, Milford, MA, USA) was kept at 35°C during the runs. Each sample ran for 15 min at a mobile phase flow rate of 1 mL/min. The injection volume and detection wavelength were 20 pL and 254 nm, respectively. Standard solutions (6.25-1000 pg/mL) were freshly prepared by serially diluting a 100 mg/mL HCQS solution aliquot that had been filtered through a sterile Millex-GP 0.22 pm hydrophilic polyethersulfone membrane syringe filter (Millipore, Burlington, MA, USA) (see below for the method). The diluent for the standard solutions was deionized water and 50:50 v/v methanokwater, depending on the diluent used for the samples.

Effect of filtration on HCQS solutions

The effect of filtration on the drug concentration, osmolality, and pH was investigated because the HCQS solutions would be sterilised by filtration before nebulisation. Approximately 3 mL of the 20, 50, or 100 mg/mL HCQS solution was drawn into a 3 mL syringe (Terumo, Tokyo, Japan). A sterile Millex-GP 0.22 pm hydrophilic polyethersulfone membrane syringe filter was then attached to the syringe. About 1 mL of the solution was ejected through the filter and discarded. The remaining 2 mL in the syringe was filtered and collected into a 2 mL microcentrifuge tube (Quality Scientific Plastics, Petaluma, CA, USA). The drug concentration, osmolality, and pH of the unfiltered and filtered solutions were measured as outlined in above. For the HPLC runs, all samples were diluted with deionised water to 500 pg/mL to be within the concentration range of the standard curve.

Recovery of HCQS from SureGard filters

SureGard filters (Bird Healthcare, Bayswater, VIC, Australia) were used in the dose output and cascade impaction runs (connected between the impactor and the vacuum pump) to collect the nebulised droplets so the recovery of HCQS from this type of filter was investigated. These filters were spiked with 2 or 75 mg of HCQS by adding 20 or 750 pL of a 100 mg/mL HCQS nebulised solution to a new filter, respectively. The openings of the filter were sealed with Parafilm (Bemis, Oshkosh, WI, USA) after adding 10 mL of deionised water or 50:50 v/v methanokwater. The filters were immediately shaken by hand for 5 minutes or left to stand for 30 minutes first, followed by 5 minutes of shaking. The samples were diluted 10-fold with deionised water or 50:50 v/v methanokwater accordingly before HPLC assay.

Dose output

The HCQS dose output from three new Aerogen^® Solo nebulisers (Mesh numbers 0901059-0822, 0901059-0797, and 0901059-1623) was measured with individualised Aerogen Ultra aerosol chambers. These nebulisers plus aerosol chambers will be referred to in this report as Nebulisers 1, 2, and 3, respectively. The same Aerogen controller was used for all experiments. One SureGard filter was connected to the outlet of the Aerogen Ultra mouthpiece. A filter was also fitted to the exhaust end of the mouthpiece and the exhaust port at the bottom of the Aerogen Ultra. There were thus one outlet filter and two exhaust filters. Silicone adaptors were used to connect the mouthpiece to the outlet and one of the exhaust filters. The experiments were conducted under ambient conditions (18-25°C, 20-65% RH).

The procedure followed the USP method (United States Pharmacopeia 43 - National Formulary 38, 2020) except that the aerosols were collected from the start to the end of nebulisation instead of collecting them for the first minute using one output filter and then collecting the rest of the aerosols with another output filter. This was to avoid drug loss when changing the filters. It would also simplify the experimental procedure. The output filter was not overloaded by the lengthening of collection duration. The end of nebulisation was determined by visual inspection when no solution remained in the nebuliser.

Nebulised dose output was measured for the following HCQS solutions: 1 mL of 20 mg/mL, 1 mL of 50 mg/mL, 1 mL of 100 mg/mL, and 1.5 mL of 20 mg/mL. The same scheme was adopted for the laser diffraction and cascade impaction experiments (see below).

HCQS solution was added into the reservoir of the Aerogen Solo by pipetting. The PWG-33 breathing simulator (Piston Medical, Budapest, Hungary) was connected to the output filter. The simulated breathing waveform was sinusoidal at 15 cycles/minute, with an inhalation-to-exhalation ratio of 1:1 and a tidal volume of 500 mL (United States Pharmacopeia 43 - National Formulary 38, 2020). The outlet and exhaust filters captured droplets exiting the nebulisers during the inhalation and exhalation phases in the breathing cycle, respectively. The nebuliser and breathing simulator were operated from the start to the end of nebulisation, after which the setup was left to stand for 20 minutes before being removed and assayed. This was to allow the droplets in the Aerogen Ultra to settle by gravitational sedimentation and avoid potential aerosol loss if the setup was disassembled immediately. The runs were conducted in triplicate for each nebuliser.

The openings of the two exhaust filters were sealed with Parafilm after adding in 10 mL of deionised water. The exhaust filters were then exhaustively rinsed by shaking for 5 minutes. The outlet filter was placed into a 600 mL glass beaker. Four hundred millilitres of deionised water, a glass weight, and magnetic stirrer were added into that beaker afterwards. The glass weight was to weigh down the filter to ensure its complete immersion in the water. The mixture was magnetically stirred for 5 minutes, followed by shaking for another 5 minutes. The liquid reservoir and outlet of the Aerogen Solo were exhaustively washed with 10 mL of deionised water and 6 minutes of shaking in total. The same was performed on the two silicone adaptors. The washings were collected into a 100 mL volumetric flask. The openings of the Aerogen Ultra were sealed with Parafilm after adding in about 10 mL of deionised water. The whole chamber was exhaustively rinsed in the same manner as described for Aerogen Solo. All washings were pooled into the same volumetric flask. The volume was made up to 100 mL with deionised water. All samples were assayed by HPLC. Laser diffraction

The nebulised droplets were sized by laser diffraction using Spraytec (Malvern Panalytical, Malvern, UK) with an inhalation cell and at an acquisition frequency of 2.5 kHz. The outlet of the Aerogen Ultra mouthpiece was positioned 1 cm from the laser measurement zone to minimise evaporation during measurement. A vacuum pump connected to the other end of the inhalation cell with entrained dilution air was used to remove the aerosols continuously to 1) prevent droplet re-entrainment of droplets into the laser measurement zone; and 2) maintain the laser signal transmission > 70% to minimise multiple scattering.

The Aerogen Ultra mouthpiece was not sealed to the inhalation cell so the airflow through the Aerogen Ultra was unknown. Signals from Detectors 1-10 were excluded to account for beam steering effects. The real and imaginary refractive indices for the droplets were taken to be the same as those for water, which were 1.33 and 0.00, respectively. The refractive index for air was 1.00. These values were deemed appropriate because all measurements showed low residual values (< 0.5%). The droplets were sized when the signal transmission was < 99%. The duration of nebulisation was the time that aerosols were seen by eye to traverse continuously through the laser measurement zone. The raw data of each run were processed to yield an averaged volumetric diameter distribution, from which the volumetric median diameter (VMD) and geometric standard deviation (GSD) were derived. The percentage of aerosol sample by volume under 1, 2, 3, 5, and 10 pm were also calculated.

Cascade impaction

The aerosol performance of the three Aerogen Solo nebulisers coupled to their respective Aerogen Ultra aerosol chambers was measured by the USP method using a Next Generation Impactor (NGI; USP Apparatus 5) without a pre-separator (United States Pharmacopeia 43 - National Formulary 38, 2020). The same Aerogen controller was used for all the experiments. The NGI and throat were chilled at 5°C for at least 90 minutes beforehand. After chilling, a SureGard filter was connected to the NGI after the micro-orifice collector (MOC) to capture any drug that passed beyond the lowest impactor stage. The sealing of the apparatus was verified before each run by a vacuum leak test, after which the airflow rate was set to 15 L/min. A silicone adaptor was used to connect the mouthpiece to the USP induction port (throat). The experiments were conducted under ambient conditions (18-25°C, 20-65% RH).

HCQS solution was added into the reservoir of the Aerogen Solo by pipetting. No exhaust filters were required to be connected to the Aerogen Ultra because the airflow was suction-only. The nebuliser and vacuum pump were operated from the start to the end of nebulisation. The end of nebulisation was determined by visual inspection when no solution remained in the nebuliser. The setup was left to stand for 20 minutes before being removed and assayed. The co-solvent used for all NGI samples was 50:50 v/v methanokwater. For the 20 mg/mL HCQS runs, the Aerogen Solo, and Aerogen Ultra were exhaustively washed with this co-solvent, collected into a 100 mL volumetric flask, and made up to volume. The post- NGI filter was washed with 10 mL of the co-solvent, as for the dose output exhaust filter. The adaptor, throat and NGI impactor stages were washed with 4 mL of the co-solvent. The assay for the 100 mg/mL HCQS runs was conducted in the same manner, except that Stages 1-6 were washed with 20 mL instead of 4 mL of the co- solvent.

The loaded dose was the amount of HCQS added into the nebuliser. The emitted dose was the total amount of drug assayed from the adaptor to the post-NGI filter. The recovered dose was the total amount of HCQS assayed on all the parts in the experimental setup, i.e. from the nebuliser to the post-NGI filter. Fine particle doses (FPDs) under 1, 2, 3, 5, and 10 pm were calculated, from which the corresponding fine particle fractions (FPFs) with respect to the loaded, emitted, and recovered doses were then derived. Likewise, the mass median aerodynamic diameter (MMAD) and GSD with respect to the recovered dose and the emitted dose were calculated. The MMAD was the diameter at 50% undersize interpolated from the cumulative recovered and emitted doses. The GSD was calculated by dividing the MMAD by the diameter at 16% undersize, which was in turn interpolated from the cumulative recovered and emitted doses.

Measurement of the density of HCQS solutions

The density of HCQS solutions (20, 50, and 100 mg/mL) was measured by first weighing deionised water in a 10 mL volumetric flask, filled to the mark. After discarding the water and drying the volumetric flask, HCQS solution was added to the mark and weighed. The density of the HCQS solutions was calculated with the following equation.

Equation 1: p_H = pw(m_H/m_w) where pn and pw are the densities of HCQS solution and deionised water, respectively; and ni_fl and niw are the masses of HCQS solution and deionised water in the 10 mL volumetric flasks, respectively. The density of deionised water at 24°C, at which the measurements were conducted, was interpolated from the water density data in the CRC Handbook of Chemistry and Physics (Chemical Rubber Company, 2020). Three volumetric flasks were used to obtain triplicate measurements for each solution. The densities of the HCQS solutions were used to convert the volumetric diameters measured by laser diffraction to a volumetric aerodynamic diameter using Equation 2:

Equation 2: d„ = d_v{pnlpo† ⁵ where d_a and d_v are aerodynamic and volumetric diameters, respectively; and po is unit density (1 g/cm³). The volumetric aerodynamic diameter was used for comparing the droplet sizes measured by laser diffraction to those by cascade impaction.

Statistical analysis

One-way analysis of variance followed by Tukey post hoc test were performed using the SPSS software (IBM, Armonk, NY, USA). Statistical differences were indicated by p < 0.05 and a < 0.05.

Effect of filtration on drug concentration, osmolality, and pH

Two batches of 100 mg/mL solutions were made (Batches A and B). Batch A was used to obtain the 20 mg/mL solution by dilution with normal saline, while Batch B was used directly for the 100 mg/mL experiments and for making the 50 mg/mL solution. The osmolality of the normal saline used for dilution to 20 mg/mL and 50 mg/mL was 284.0 ± 3.2 and 287.0 ± 7.8 mOsmol/kg H2O, respectively ( n = 4). No drug degradation was observed over the 15 days during which all the experiments were performed. The drug concentration, osmolality, and pH of the HCQS solutions before and after filtration are presented in Table 3.

Table 3. Drug concentration, osmolality, and pH of HCQS solutions before and after filtration.

Osmolality is presented as mean ± standard deviation (n = 4). One pH measurement was made for each solution (n = 1).

The osmolality and pH of all solutions were within the target ranges, regardless of filtration. The five-fold dilution of the Batch A 100 mg/mL solution to 20 mg/mL reduced the osmolality from 323.0 to 286.5, which was close to that of normal saline (284.0 mOsmol/kg H2O). HCQS concentration was not affected by filtration. On the other hand, osmolality and pH decreased after filtration but the difference was not significant. Similar trends were observed for the Batch B 100 mg/mL and 50 mg/mL solutions. The osmolality and pH of the 50 mg/mL were between those of the 20 mg/mL and 100 mg/mL solutions.

The retention time of the HCQS peak in the HPLC chromatogram was about 8 minutes. Table 3 shows the regression equations of the calibration curves with the mean slopes and y-intercept. They were obtained using fresh standard solutions over 11 and 14 days with deionised water and 50:50 v/v methanohwater as the diluent, respectively. The standard curves were similar between the days and were linear (r² ~ 1) from 6.25-1000 pg/mL. The detection and quantitation limits were derived by Equations 3 and 4, respectively (Guidance for Industry - Q2B Validation of Analytical Procedures: Methodology, 1996). The values of slope featured in these equations were taken to be the mean slopes shown in Table 4.

Equation 3: Detection limit = (3.3 x Standard deviation of the y-intercepts)/Slope Equation 4: Quantitation limit = (10 x Standard deviation of the y-intercepts)/Slope The HPLC method was more sensitive with deionised water as the diluent, as shown by the lower detection and quantitation limits (Table 4).

Table 4. HPLC calibration curves, detection limit, and quantitation limit for HCQS from 6.25-1000 pg/mL.

Regression equation Detection limit Quantitation limit

(pg/mL) (pg/mL)

Deionised water A = 43294C + 32746 2.7 8.3

50:50 v/v methanohwater A = 42848C - 19037 7.2 21.7 A = Peak area at 254 nm.

C = HCQS concentration in pg/mL.

The slopes and y-intercepts were the mean of 11 and 14 values for water and the co- solvent, respectively. Recovery of HCQS from SureGard filters

The recovery of HCQS from the spiked SureGard filters using deionised water and 50:50 v/v methanohwater is presented in Table 5. Deionised water was more efficient than the co-solvent for extracting HCQS from the filters. It obviated the need for the 30-minute standing time to allow the filter to soak before shaking. Drug adsorption to the filter was appreciable at the low spiked drug level, as 4-5% of drug could not be recovered even when water was used. This was even more significant (11-12%) with the co-solvent. Table 5. Recovery of HCQS from spiked SureGard filters.

Deionised water 50:50 v/v methanol: water

2 mg HCQS Immediate 5 min shaking 95.7% 87.3%

30 min standing, 5 min shaking 95.3% 88.9%

75 mg 100.7% 94.4%

Immediate 5 min shaking

HCQS

30 min standing, 5 min shaking 100.2% 99.3%

One measurement was performed for each scenario (n 1).

Dose output

The nebulisation duration of the 1 mL loaded dose runs is shown in Table 6.

Table 6. Nebulisation duration of the dose output experiments.

Nebuliser 1 Nebuliser 2 Nebuliser 3 All nebulisers

1 mL, 20 mg/mL 3 min 26 s ± 7 s 4 min 27 s ± 31 s 3 min 41 s ± 18 s 3 min 51 s ± 33 s

1 mL, 50 mg/mL 5 min 4 s ± 13 s 5 min 7 s ± 10 s 5 min 8 s ± 16 s 5 min 6 s ± 11 s

1 mL, 100 6 min 24 s ± 26 s 6 min 25 s ± 35 s 6 min 22 s ± 6 s 6 min 24 s ± 22 s mg/mL 1.5 mL, 100

10 min 55 s ± 43 s 9 min 31 s ± 36 s 10 min 24 s ± 45 s 10 min 17 s ± 52 s mg/mL

Data presented as mean ± standard deviation (n = 3 for Nebulisers 1, 2, and 3; n = 9 for all nebulisers).

However, the correlation between solute concentration and nebulisation duration was non-linear. The nebulisation duration with 1.5 mL of 100 mg/mL was understandably longer than its 1 mL counterpart.

The recovered dose for the runs was generally 95-105% of the loaded dose so drug recovery was satisfactory. The absolute and relative doses with respect to the recovered dose for the various parts of the experimental setup are shown in Figure 8. The data for the absolute doses showed that the amount of drug reaching the exhaust filters was very low.

Most of the drug was shared between the output filter and the Aerogen Solo/Ultra. The output dose was approximately proportional to the loaded dose. This was confirmed by the similar distributions of relative doses on the various parts of the experimental setup between the concentration/volume combinations. The amount of drug that exited the nebuliser setup during the exhalation phase of the breathing cycle (i.e. those collected on the two exhaust filters) was low, with < 2% of the recovered dose on each filter. About half of the recovered dose was emitted onto the output filter, which represented the amount of HCQS that a patient would inhale assuming that the simulated breathing cycle is representative of the patient’s breathing. The remainder was retained in the Aerogen Solo/Ultra. The output with respect to the recovered dose from 1 mL of 50 mg/mL was slightly lower than that from 1.5 mL of 100 mg/mL (Figure 8). This is explained by the correspondingly higher drug retention in the Aerogen Solo/Ultra.

Laser diffraction

The droplet size distributions measured by laser diffraction were stable over the entire measurement period for each run. The nebulisation duration was longer than the actual measurement time because the aerosol concentrations were low (“thin” aerosols) at the start and end of nebulisation so the laser signal transmission at these times were higher than the trigger threshold for measurement (99.9%). The measurements generally started a few seconds after aerosols appeared in the measurement zone for all drug concentrations/volumes. The thin aerosol tailing near the end of nebulisation (i.e. thin aerosols in the measurement zone but no sizing was triggered) took about 30 seconds and was particularly longer (up to 1 minute) for 20 mg/mL.

The size distributions were all monomodal and reproducible between the three nebulisers, with the peak at about 5 pm. There was a slight shift in the distribution to the smaller size between 100 mg/mL (both volumes) and the other two HCQS concentrations. This difference was more obvious in the VMD (Figure 9). Although the VMD for all concentrations/volumes was between 4.3-5.2 pm, the droplets produced from 100 mg/mL solutions were slightly but significantly (p<0.05) smaller than those from 20 and 50 mg/mL (Figure 9). The GSD was relatively consistent between the four concentrations/volumes, at about 1.8 (Figure 10). However, the GSD from 1.5 mL of 100 mg/mL was also slightly but significantly (p<0.05) lower than that from 1 mL of 20 mg/mL.

The percentage of aerosol sample by volume under 1, 2, 3, 5, and 10 pm is shown in Figure 11. About 50-60% of the aerosols was < 5 pm. The nebulisers produced minimal submicron droplets at all concentrations/volumes but the 100 mg/mL solution consistently produced more droplets by volume than 20 and 50 mg/mL at all cut-off diameters. In other words, the droplets from the 100 mg/mL solution were smaller than those from the other two solutions. No clear dependence between droplet size and relative humidity was observed so the difference in droplet size was attributed to the solute concentration and the resultant changes in the physicochemical characteristics of the solutions.

Cascade impaction

The nebulisation durations of the NGI runs (Table 7) were similar to those for the dose output runs (Table 6).

Table 7. Nebulisation duration of the cascade impaction experiments.

Nebuliser 1 Nebuliser 2 Nebuliser 3 All nebulisers

1 mL, 20 mg/mL 3 min 48 s ± 7 s 3 min 50 s ± 44 s 3 min 32 s ± 22 s 3 min 44 s ± 26 s

1 mL, 50 mg/mL 5 min 43 s ± 9 s 5 min 15 s ± 19 s 5 min 35 s ± 10 s 5 min 31 s ± 17 s

1 mL, 100 6 min 41 s ± 42 s 6 min 18 s ± 21 s

6 min 26 s ± 33 s 6 min 28 s ± 30 s mg/mL

1.5 mL, 100

10 min 48 s ± 20 s 9 min 53 s ± 48 s 9 min 6 s ± 26 s 9 min 56 s ± 53 s mg/mL

Data presented as mean ± standard deviation (n = 3 for Nebulisers 1, 2, and 3; n = 9 for all nebulisers). The recovered dose was close to the loaded dose for all the runs so drug recovery was satisfactory. The absolute and relative doses (with respect to the recovered dose) for the various parts of the setup are shown in Figure 12. Only a small amount of HCQS (< 1%) was collected on the post-NGI filter so the NGI captured practically all the emitted doses. About 30-40% of the recovered dose remained in the nebulisers after the NGI runs, compared to 50% after the dose output runs (Figures 12 and 8, respectively). This might be due to the vacuum pump continuously removing droplets from the Aerogen Ultra into the NGI rather than blowing them back repeatedly into the aerosol chamber, as in the case of the breath simulator during the exhalation phase employed in the delivered dose experiments. The overall aerosol performance profiles were similar between the concentrations/volumes, with minimal throat deposition (Figure 12). However, 1.5 mL of 100 mg/mL showed more drug on Stages 4 and 5 so there was a higher proportion of fine droplets.

The emitted dose, FPD, FPF < 5 pm loaded, FPF < 5 pm emitted, MMAD emitted, and GSD emitted derived from the NGI data are summarised in Table 8, together with other key parameters measured in the dose output and laser diffraction experiments for comparison. Table 8. Summary of the key parameters measured in the dose output, laser diffraction, and cascade impaction experiments. l mF of 20 l mF of 50 l mF of lOO 1.5 mF of 100 mg/mF mg/mF mg/mF mg/mF

Dose output

Dose collected in 9.05 + 0.96 21.67 + 2.81 48.76 + 5.57 75.88 + 5.87 output filter (mg)

Faser diffraction

VMD (mih) 4.95 + 0.17 5.19 + 0.25 4.34 + 0.31 4.42 + 0.19

GSD 1.85 ± 0.04 1.84 + 0.02 1.83 + 0.04 1.80 + 0.02

Cascade impaction

Emitted dose (mg) 13.2 + 1.15 33.8 + 2.19 61.9 + 5.28 99.0 + 5.71

FPD < 5 pm (mg) 9.49 + 1.13 22.7 + 1.34 44.2 + 5.60 81.6 + 6.93

FPF < 5 pm loaded (%) 47.3 + 5.64 45.3 + 2.74 44.2 + 5.60 54.4 + 4.57

FPF < 5 pm emitted 71.8 + 3.44 67.3 + 3.25 71.3 + 4.71 82.3 + 3.02

(%)

MMAD emitted (pm) 3.00 + 0.18 3.27 + 0.25 2.99 + 0.27 2.50 + 0.17

GSD emitted 2.02 + 0.06 1.94 + 0.14 1.88 + 0.05 1.75 + 0.05

Data presented as mean + standard deviation in = 9). The higher FPF and MMAD of 1.5 mL of 100 mg/mF also indicate that it produced smaller droplets than the other concentrations/volumes. The GSD gradually decreased with increasing concentration/volume so the size distribution became narrower. These trends were also observed in the VMD and GSD in the laser diffraction data (Table 8). The doses collected in the output filter in the dose output experiments were consistently lower than the emitted doses in cascade impaction. This was because the vacuum pump in the latter constantly pulled the aerosol out of the nebuliser (continuous “inhalation”) and the breath simulator in the former generated a sinusoidal flow (periodic “inhalation” and “exhalation”). Comparison of dose output, laser diffraction, and cascade impaction data

Faser diffraction and cascade impaction data were compared to check their correlation. To improve the accuracy of the comparison, the major parameters from laser diffraction (VMD and %V < 1, 2, 3, 5, 10 pm) were converted to their volumetric aerodynamic diameters by Equation 2. The density of the 20, 50, and 100 mg/mF HCQS solutions were measured to be 1.008, 1.019, and 1.033 g/cm³, respectively, and were used in the calculation. The data are shown in Table 9.

Table 9. Comparison of cascade impaction and laser diffraction data. 1 mL of 20 mg/mL

1 mL of 50 mg/mL

Cascade impaction data Laser diffraction data Cascade impaction

(Aerodynamic diamete data / Laser diffraction data (%)

FPF emitted < 1 4.96 %V < 1 pm 1.05 + 0.30 506.31 + 166.86 pm (%) 0.30

FPF emitted < 2 24.4 %V < 2 pm 8.63 + 0.67 286.29 + 46.16 pm (%) 0.67

FPF emitted < 3 45.2 %V < 3 pm 21.46 + 1.73 212.77 + 28.92 pm (%) 1.73

Correlation between the two techniques was reflected in the percent ratio of each parameter, which was the quotient of a given parameter measured by cascade impaction and that by laser diffraction. The FPFs measured by cascade impaction for all concentrations/volumes were consistently higher than those by laser diffraction at the corresponding cut-off diameters (Table 9). By the same token, the MMAD with respect to the emitted dose measured by cascade impaction was smaller than the volumetric median aerodynamic diameter (VMAD) by laser diffraction. The smaller particle sizes measured by cascade impaction could be attributed to droplet evaporation in the NGI. Despite this, its width remained relatively constant. The deviation between the corresponding GSDs was 97- 110%, indicating that evaporation in the NGI was a monotonous shift to the smaller sizes without changing the width of the distribution. The main trend observed in the FPFs and %V undersize was that the lower the cutoff diameter the larger the deviation between the two datasets, with relatively close agreement at 10 pm (97-104% deviation), to > 120% deviation at 5 pm, and > 200% deviation at 2 pm. This was most likely due to the faster evaporation rates of small droplets, which increased the FPF to a greater extent at the lower cutoff sizes.

In addition, the deviation between the two datasets decreased with increasing HCQS concentration for the 1 mL solutions. This might be due to the reduction in vapour pressure with increasing HCQS concentration, which decreased the evaporation rate. Droplet size was observed to decrease with increasing HCQS concentration, especially between 50 and 100 mg/mL (Tables 8 and 9). An increase in the concentration of ionic species increased the electrical conductivity of the liquid, which then dissipated the high charges that would otherwise be present between water and the nebuliser mesh.

Consequently, fluid adhesion to the mesh was reduced and droplets could be detached easier, resulting in the production of smaller droplets. However, the reduction in droplet size with increasing ionic concentration is sigmoidal. In other words, the droplet size will reach a plateau after the ionic concentration exceeds a threshold. The threshold concentration is dependent on the ionic species and liquid vehicle and beyond which other physicochemical factors ( e.g . viscosity and surface tension) may then become dominant in affecting droplet size.

The in vivo antiviral and anti-inflammatory concentrations of hydroxychloroquine in the airways is unknown but its in vitro antiviral EC 50 is approximately 1-5 mM. Idkaidek et al employed a physiologically-based pharmacokinetic model to estimate the inhaled dose needed for COVID-19 based on this concentration range ( Drug Res (Stuttg), December 2020). The model featured droplets with a VMD of 5.6 pm. The proportions depositing in the trachea, bronchioles, and alveoli were 10, 13, and 30% by mass, respectively. Their sum (53%) could be interpreted as the proportion of the emitted aerosol < 5 pm because they theoretically deposited in the lungs. It was found that inhaling 25 mg hydroxychloroquine twice a day could achieve a maximum concentration (C_max) ³ 7 pM in the various parts of the lungs, while the plasma C_max was only 0.18 pM. If the inhaled dose was doubled to 50 mg hydroxychloroquine twice a day, then the lung Cmax reached > 13 pM and plasma Cmax increased to 0.35 pM. Thus, pulmonary drug concentrations higher than the in vitro antiviral EC 50 with low systemic absorption is potentially achievable. The plasma hydroxychloroquine concentration for rheumatoid arthritis treatment is typically < 1 pM, while serious toxicity was associated with plasma levels from 2.05-18.16 pM ( Jordan, P., Brookes, J.G., Nikolic, G., Le Couteur, D.G., 1999. Hydroxychloroquine overdose: Toxicokinetics and management. Clinical Toxicology 37, 861-864). Therefore, systemic adverse effects should be minimal with the low plasma concentrations from the inhalation regimens outlined above. The emitted dose obtained from the dose output experiments were 9.1-75.9 mg (Table 8), depending on the concentration/volume of the HCQS solution. This encompassed the proposed range of 25- 50 mg hydroxychloroquine (equivalent to 32.3-64.5 mg HCQS). The VMD of HCQS droplets discussed herein were 4.3-5.2 pm, with 50-60% of the them < 5 pm (Table 8, Figure 11). This suggests that if our HCQS aerosols were inhaled twice a day, especially with the two volumes of 100 mg/mL HCQS solutions (dose outputs of 48.8-75.9 mg), they would be able to produce pulmonary drug concentrations above the in vitro antiviral EC50. To put this into perspective, 400-600 mg of HCQS were delivered orally per day in previous COVID-19 clinical trials (Pastick et al, 2020). This is 5- 12-fold higher than the emitted doses from the two volumes of 100 mg/mL HCQS solutions. Inhalation would be more efficient and safer for potential treatment of COVID-19. Example 5. Assessment of the pharmacokinetic profile of hydroxychloroquine after single intravenous, oral and intratracheal instillation in male Sprague Dawley rats.

The aim of this study was to evaluate overall systemic levels, and specifically heart and lung exposure levels, after systemic and local dosing of hydroxychloroquine sulfate with the aim to establish safety benefits using topical, intra-tracheal (IT) administration compared to systemic administration (PO and IV). Tissue levels for heart and lung were assessed, and full PK profiles for plasma and whole blood were generated. The lungs were separated into parenchyma and large airways (including trachea). The concentration of hydroxychloroquine in these separated tissues was independently assessed. The study design is shown in the following table.

Figure 15 depicts plasma concentrations of HCQ in the animals following administration, and Figure 16 depicts blood concentrations of HCQ in the animals following administration. Figure 17 depicts tissue concentrations of HCQ in the animals following administration.

Example 6. Dose Selection and Pharmacokinetic Study

Orally administered hydroxychloroquine sulfate (HCQ) has produced negative results as a treatment for COVID-19, despite reported antiviral activity in vitro (IC50 = 0.7-119 mM). Here it is demonstrated that low doses of aerosolized HCQ (aHCQ) safely and rapidly achieve high respiratory tissue concentrations while minimizing systemic toxicity. As a prelude to the human study, studies were performed comparing HCQ’s blood and tissue pharmacokinetics after administration to rats by 3 different routes - IV, oral and intra- tracheal. An intra-tracheal dose of 0.18 mg/kg, less than 14% of the oral dose, was selected based on allometric scaling between species accounting for differences in delivery efficiency and hmg-to-body weight ratio. As shown in Fig. 13, compared to oral administration (PO), intratracheal instillation (IT), yielded a smaller blood area under the curve, mean peak lung concentrations that surpassed the highest reported IC50 for SARS-CoV-2 and were not achieved with oral administration, and minimal cardiac tissue exposure, the organ at greatest risk for adverse events, compared to oral and IV administration.

In the Phase 1 study, aHCQ was administered to 2 sentinel healthy volunteers at 20 mg. After safety review, 50 mg was administered to 2 sentinels. Again, after safety review, 6 healthy volunteers were randomized in a double-blind manner to single dose aHCQ 50 mg or placebo.

Study outcomes included clinical assessments, pulmonary function tests, ECGs, pharmacokinetics, and participant-reported outcomes. Measures to ensure the environmental safety during the pandemic included using the Aerogen nebulizer, which has a low level of emitted aerosols, and performing aerosol generating procedures under airborne precautions, and only after participants tested negative for SARS-CoV-2. There were 10 study participants (4 male and 6 female) with a mean age of 55 years ± 13 years. The test formulation was aerosolized HCQ sulfate (100 mg/ml). The placebo was sodium chloride inhalation solution (USP, 0.9%).

As shown in Fig. 14, aHCQ produced a rapid but short-lived peak in blood concentration followed by gradual decline. The Area Under the Curve was less than 15% of values reported after oral HCQ (200 mg) from published data. Based on the observed blood/tissue ratio observed in rats, the epithelial lining fluid concentration achieved immediately after administration of aHCQ, based on regional aerosol deposition patterns, was predicted to be more than 2,000 mM, but this concentration was also predicted to drop rapidly. To predict the tissue concentrations achieved after aHCQ administration, blood PK in humans was correlated with observed tissue to blood ratio of HCQ in rats after intratracheal instillation. These data suggest that it is possible to achieve respiratory concentrations sufficient to inhibit SARS-CoV-2 without cardiac toxicity. As such, it is shown that administering aHCQ at a fraction of the usual oral dose is likely to rapidly achieve respiratory tract concentrations sufficient to inhibit SARS-CoV-2 in vitro.

Claims

1. A pharmaceutical composition for inhalation, comprising a compound selected from the group consisting of hydroxychloroquine, primaquine, mepacrine, mefloquine, pamaquine, pentaquine, tafenoquine, lumefantrine, pyronaridine, and salts thereof; and a carrier or diluent.

2. The composition of claim 1, further comprising one or more of: a tonicity agent, a flavoring agent, a preservative, a surfactant, a stabilizer, a pH adjustment agent, and a propellant.

3. The composition of any one of the preceding claims, wherein the composition is formulated for inhalation by mouth or nose.

4. The composition of any one of the preceding claims, wherein the compound is hydroxychloroquine, or a pharmaceutically acceptable salt thereof.

5. The composition of claim 4, wherein the compound is (R)-hydroxychloroquine, or pharmaceutically acceptable salt thereof.

6. The composition of claim 4, wherein the compound is (S)-hydroxychloroquine, or pharmaceutically acceptable salt thereof.

7. The composition of any one of the preceding claims, wherein the compound is a sulfate salt.

8. The composition of claim 7, wherein the compound is a monosulfate salt.

9. The composition of any one of the preceding claims, wherein the composition is a dry powder.

10. The composition of claim 9, wherein the weight percent of compound in the composition is at least 80%, at least 90%, at least 95%, or 100%.

11. The composition of any one of claims 9-10, wherein the dry powder is formulated for direct inhalation.

12. The composition of any one of claims 9-11, wherein the dry powder is formulated for reconstitution as a solution.

13. The composition of any one of claims 1-8, wherein the carrier is a liquid carrier.

14. The composition of claim 13, wherein the composition is a solution, microemulsion, suspension, or co-suspension.

15. The composition of claim 14, wherein the composition is formulated for aerosolization.

16. The composition of any one of the preceding claims, wherein the compound is present in the form of a microparticle, nanoparticle, cyclodextrin complex, liposome, or noisome.

17. The composition of any one of the preceding claims, wherein the composition is formulated to suppress flavor and/or irritation.

18. The composition of any one of claims 13-17, wherein the concentration of the compound in the composition is in the range of 0.01-lM.

19. The composition of any one of claims 13-18, wherein the concentration of the compound in the composition is in the range of 5mg/ml to 100 mg/ml.

20. The composition of any one of claims 11-16, wherein the liquid carrier comprises water, saline, ethanol, or a combination thereof.

21. The composition of claim 20, wherein the water is deionized, microfiltered, and or sterilized water.

22. The composition of any one of the preceding claims, wherein the flavoring agent comprises a GRAS (generally recognized as safe) material.

23. The composition of any one of the preceding claims comprising a flavoring agent, wherein the flavoring agent comprises a terpene.

24. The composition of any one of the preceding claims, wherein the favoring agent is a plant extract.

25. The composition of any one of the preceding claims, wherein the concentration of the flavoring agent is in the range of 0.001M-1M.

26. The composition of any one of the preceding claims comprising a preservative, wherein the preservative inhibits decomposition of the compound, or prevents bacterial growth.

27. The composition of any one of the preceding claims, wherein the preservative is an antioxidant.

28. The composition of claim 27, wherein the antioxidant is a non-ionic organic molecule, or an inorganic or organic ionic molecule, or a salt thereof.

29. The composition of any one of the preceding claims, wherein the preservative is a hydroxylated organic molecule.

30. The composition of claim 29, wherein the hydroxylated organic molecule is a citrate, phenol, or phenol derivative.

31. The composition of any one of the preceding claims comprising a surfactant, wherein the surfactant is an ionic or non-ionic surfactant.

32. The composition of any one of the preceding claims comprising a stabilizer, wherein the stabilizer is an ionic or non-ionic stabilizer.

33. The composition of any one of the preceding claims comprising a propellant, wherein the propellant is a gas, or a halogenated or nonhalogenated hydrodrocarbon.

34. The composition of claim 33, wherein the propellant is carbon dioxide, propane, butane, or isobutane.

35. The composition of claim 32, wherein the propellant is a chlorofluorocarbon, or a fluorocarbon.

36. The composition of any one of the preceding claims, comprising a pH adjustment agent.

37. The composition of claim 36, wherein the pH adjustment agent is an acid or a base ( e.g ., hydrochloric acid or sodium hydroxide).

38. The composition of any one of the preceding claims, wherein the pH of the composition is in the range of 6.0 to 7.8.

39. The composition of claim 38, wherein the pH of the composition is in the range of 7.0 to 7.4.

40. The composition of any one of the preceding claims, wherein the compound is a mixed salt.

41. The composition of any one of the preceding claims, wherein the composition is hypotonic, isotonic, or hypertonic.

42. The composition of any one of claims 13-41, wherein the viscosity of the composition is in the range of 0.8- 1.6 Pa-s.

43. The composition of any one of any one of claims 13-42, wherein the osmolality of the composition is in the range of 100-1000 mmol/kg.

44. The composition of any one of the preceding claims, further comprising one or more additional therapeutic agents.

45. The composition of claim 44, wherein the one or more additional therapeutic agents are independently an anti-viral, an anti-coagulant, an anti-parasitic, an anti-microbial, an anesthetic, a bronchodilator, a steroid, anti-inflammatory or a vitamin.

46. The composition of any one of the preceding claims, wherein the composition is stable at 25 °C for at least 15 days, at least 30 days, or at least one year.

47. The composition of any one of the preceding claims, wherein the composition is stable at 40 °C for at least 15 days, at least 30 days, or at least one year.

48. The composition of any one of the preceding claims, for use in treating or preventing a viral infection in a subject.

49. The composition of claim 48, wherein the viral infection is an infection of a human rhinovirus or a coronavirus in the subject.

50. The composition of any one of claims 1-49, wherein the subject is human.

51. The composition of any one of claims 1-49, wherein the subject is a non-human animal.

52. A kit for preparing a composition of any one of the preceding claims, comprising the composition components and instructions for preparing the composition.

53. An aerosol comprising the pharmaceutical composition of any one of claims 1-51.

54. A device for delivering to a subject via inhalation or exhalation ( e.g ., oral or nasal) an aerosol according to claim 53.

55. The device of claim 54, wherein the device is a nebulizer (e.g., jet, mesh, or ultrasonic), atomizer, vaporizer, or electrospray.

56. The device of any one of claims 54-55, wherein the device is propellant-driven, breath-actuated, or pump-actuated.

57. The device of any one of claims 54-56, further comprising a metering valve.

58. The device of any one of claims 54-57, wherein the device is configured to produce an aerosol of claim 53.

59. The device of claim 58, wherein the device is configured to control or regulate aerosol droplet or particle size.

60. The device of any one of claims 54-59, wherein the device is configured to produce the aerosol for a duration in the range of 5 seconds to 30 minutes.

61. The device of any one of claims 54-60, wherein the device is configured to control or regulate aerosol velocity.

62. The device of any one of claims 54-61, further comprising a filter, wherein the filter is positioned to receive the subject’s exhalation.

63. The device of claim 62, wherein the filter is a HEPA filter.

64. The device of any one of claims 54-63, wherein the device is configured to deliver an amount of the composition in the range of 1-130 mg.

65. The device of any one of claims 51-58, wherein the device is configured to deliver a dose of compound in the range of 1-130 mg.

66. The device of any one of claims 54-65, wherein the device is configured to deliver the composition to the respiratory tract with an efficiency (i.e., percent compound delivered) of at least 15%, at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%.

67. The device of any one of claims 54-66, comprising a non-pressurized reservoir containing the composition.

68. The device of any one of claims 54-67, comprising a pressurized reservoir containing the composition.

69. A method of treating or preventing a viral infection in a subject, comprising administering to the subject via inhalation an aerosol of claim 53.

70. The method of claim 69, wherein the viral infection is an infection of a human rhinovirus or a coronavirus in the subject.

71. The method of claim 70, wherein the coronavirus is SARS-CoV-2.

72. The method of any one of claims 69-71, comprising administering an amount of aerosol containing a dose of the compound in the range of 1-300 mg.

73. The method of any one of claims 69-72, comprising administering the aerosol to the subject 2 or more times, up to a maximum daily compound dosage of 800 mg.

74. The method of any one of claims 69-73, wherein the administration comprises a single inhalation of the maximum amount of aerosol that is tolerated by the subject, up to the maximum daily dose of 800 mg.

75. The method of any one of claims 69-73, wherein the administration comprises multiple inhalations of the maximum amount of aerosol that is tolerated by the subject, up to the maximum daily dose of 800 mg.

76. The method of claim 75, wherein the number of inhalations per day is 1-8, 1-4, or 4.

77. The method of any one of claims 69-76, wherein treating the viral infection comprises reducing viral count and/or relieving clinical symptoms such as fever, chills, and pain.

78. The method of claim 77, wherein the viral load of the subject is reduced by at least 95% within 24 hours, at least 95% within 2 days, at least 95% within 3 days, at least 95% within 4 days, at least 95% within 5 days, at least 95% within 6 days, at least 95% within 7 days, at least 95% within 8 days, at least 95% within 9 days, or at least 95% within 10 days.

79. The method of any one of claims 69-78, wherein the aerosol is delivered to the lungs of the subject with an efficiency in the range of 10-70%.

80. The method of any one of claims 69-78, wherein the subject is a human.

81. The method of any one of claims 69-78, wherein the subject is a non-human animal.

82. The method of any one of claims 69-81, wherein the subject has a pre-existing condition or comorbidity, e.g., the subject has or has a history of smoking, obstructive pulmonary/airway disorder, or immunodeficiency.

83. The method of claim 82, wherein the subject is receiving or has received treatment of a pre-existing condition or comorbidity.

84. The method of any one of claims 69-83, wherein treatment or prevention begins prior to the onset of symptoms in the subject.

85. The method of any one of claims 69-83, wherein treatment begins concurrently with the onset of symptoms.

86. The method of any one of claims 69-83, wherein treatment begins after the onset of symptoms (e.g., 1 hour after, 12 hours after, 24 hours after, 2-10 days after).

87. A method of preparing a composition according to any one of claims 1-49, comprising the step of selecting an acid addition salt of the compound, and optionally selecting one or more pH adjustment agents, such that the pH of the composition is in the range of 6.0-7.8, e.g., 1-1 A.