WO2023138670A1

WO2023138670A1 - Dual targeting powder formulation of antiviral agent for nasal and lung deposition through single intranasal administration

Info

Publication number: WO2023138670A1
Application number: PCT/CN2023/073248
Authority: WO
Inventors: Ka Wing Jenny LAM; Han Cong SEOW; Qiuying Liao
Original assignee: University of Hong Kong HKU
Current assignee: University of Hong Kong HKU
Priority date: 2022-01-21
Filing date: 2023-01-19
Publication date: 2023-07-27
Anticipated expiration: 2024-07-21
Also published as: CN118695855A

Abstract

A novel dual targeting powder formulation with bimodal particle size distribution. The formulation targets both the nasal cavity and the lung by a single intranasal administration. The compositions can comprise a therapeutic agent, including tamibarotene, a retinoid derivative with broad-spectrum antiviral activity. Spray freeze drying (SFD) and spray drying (SD) techniques are employed to produce tamibarotene powder formulations with specific particle size using appropriate atomizing nozzles. Two formulations of different particle size are mixed to produce a single powder formulation with dual deposition characteristic in both the nasal and lung regions. The ratio of the aerosol deposition fractions in the nasal cavity and the deep lung region can be modified by varying the mixing ratio of SD tamibarotene powder.

Description

DUAL TARGETING POWDER FORMULATION OF ANTIVIRAL AGENT FOR NASAL AND LUNG DEPOSITION THROUGH SINGLE INTRANASAL ADMINISTRATION

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 63/267,027, filed January 21, 2022, which is hereby incorporated by reference in its entirety including any tables, figures, or drawings.

BACKGROUND OF THE INVENTION

Airborne viruses are serious global threats that cause a multitude of respiratory diseases. Influenza, rhinovirus, adenovirus, enterovirus, and coronavirus are common causes of viral respiratory infections, which are highly contagious and potentially deadly. The new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) most recently caused the Coronavirus disease 2019 (COVID-19) global pandemic due to its rapid spread and devastating impact (Platto et al., 2020) . Compared to other viruses, the unprecedented spread of SARS-CoV-2 can be attributed to high viral shedding in the upper respiratory tract of asymptomatic or pre-symptomatic individuals at an early stage of infection (Wu et al., 2021) . The transmission of SARS-CoV-2 through airborne respiratory droplets and aerosols (Liu et al., 2020) highlights the importance of developing new methods that target the entire respiratory tract for preventing transmission, contraction, and rapid treatment of COVID-19.

In attempt to control the pandemic, numerous vaccines including nasal spray vaccines are being investigated to curb viral spread by inducing immunity in the airways (Zhao et al., 2020) . New therapeutic agents such as fusion inhibitors (de Vries et al., 2021) , acidic electrolyzed water (Takeda et al., 2020) and nitric oxide (Winchester et al., 2021) have been researched to directly target the virus present in the nasal cavity. Our group recently developed a dry powder formulation of tamibarotene that can potentially be repurposed for the treatment of COVID-19 by pulmonary delivery (Liao et al., 2021) . Tamibarotene is a retinoid derivative that triggers lipid metabolic reprogramming by interacting with the sterol regulatory element binding protein (SREBP) in host cells (Yuan et al., 2019) . The suppression of lipogenesis results in reduced double-membrane vesicles and viral protein palmitoylation (Yuan et al., 2020) , thereby interfering with viral entry and multiplication of a broad-spectrum of viruses including SARS-CoV-2, MERS-CoV and Influenza A (Liao et al., 2021) . Pulmonary delivery of tamibarotene reduces systemic exposure and increases drug concentration in the lung, increasing its efficacy as a broad-spectrum antiviral agent. To further develop and improve the therapeutic use of repurposed tamibarotene powder, we explore targeted delivery of tamibarotene to multiple infected sites for the treatment of respiratory viral infections.

In the case of COVID-19, spike proteins on the SARS-CoV-2 viral membrane facilitate viral entry into the host cell (Ou et al., 2020) and have strong affinity to Angiotensin-Converting Enzyme 2 (ACE2) receptors that are highly expressed on respiratory cells (Shang et al., 2020) . As ACE2 receptors are found to be expressed highest in the nasal cavity with decreasing expression from the upper to lower respiratory tract, the infectivity of SARS-CoV-2 also gradually decreases from the proximal to distal respiratory tract, suggesting that the nasal epithelium is the origin of infection and acts as a viral reservoir that subsequently spreads the virus across the respiratory mucosa by aspiration-mediated virus seeding (Hou et al., 2020) . Moreover, aerosolized droplets containing SARS-CoV-2 virus may also directly enter the deep lung region causing consequent severe infection, cytokine storm and acute respiratory distress syndrome (Salian et al., 2021) . Hence, targeting both the upper and lower respiratory tract is critical for the treatment of COVID-19 infections (Sungnak et al., 2020) . Coinfections with other viruses such as influenza also further complicates the disease, with interferon-driven upregulation of ACE2 in nasal epithelia and lung tissue increasing the susceptibility of the respiratory tract to SARS-CoV-2 infection (Ziegler et al., 2020) .

Considering the unpredictability of viral pandemics, early administration of a broad-spectrum antiviral agents that target both the upper and lower respiratory tract can be an effective strategy for the treatment and control of viral infections and co-infections.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to a dry powder formulation that can target both upper and lower respiratory tract through a single administration via the intranasal route. This can be achieved by a formulation containing particles with bimodal distribution. Particles within the aerodynamic diameter range of 1 to 5 μm are considered suitable for deposition in the lungs (fine particle fraction, FPF) while particles above 10 μm are deposited in the nasal cavity (nasal fraction, NF) . On the contrary, particles between 5 and 10 μm are usually impacted and deposited in the oropharynx region (throat fraction, TF) and eventually being swallowed. Therefore, the subject compositions maximize the FPF and NF while minimizing the TF. Dry powder for intranasal administration is developed for administration of antiviral agents due to its stability, ease of handling, and portability with the possibility of self-administration.

Two particle engineering techniques, namely spray drying (SD) and spray freeze drying (SFD) , can be used to produce the dry powder formulation due to the good aerosol performance of the particles. Both methods involve the atomization of feed liquid. In certain embodiments, the type of nozzle used for atomization can control the size of droplets, which in turn affects the aerodynamic diameter of the dry particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1B. Volumetric particle size distribution of tamibarotene powder formulations prepared by (FIG. 1A) spray freeze drying (SFD) and (FIG. 1B) spray drying (SD) . The powders were dispersed using a nasal powder device and evaluated by laser diffraction. For each formulation, the most representative volumetric particle size distribution data were plotted for comparison.

FIG. 2. Scanning electron microscopy (SEM) images of tamibarotene powder formulations prepared by spray freeze drying (SFD) and spray drying (SD) . Unformulated tamibarotene was included for comparison. The SEM images were observed under × 2, 500 magnification, scale bar = 20 μm; TFN –two-fluid nozzle; US –ultrasonic nozzle.

FIG. 3. Scanning electron microscopy (SEM) images of tamibarotene powder formulations prepared by spray freeze drying (SFD) and spray drying (SD) . Unformulated tamibarotene was included for comparison. The SEM images were observed under × 500 magnification, scale bar =100 μm; TFN –two-fluid nozzle; US –ultrasonic nozzle.

FIGs. 4A-4D. Aerosol performance of tamibarotene powder formulations dispersed from nasal device operated at a flow rate of 28.3 L/min. The formulations were evaluated by Next Generation Impactor (NGI) coupled with 1 L glass expansion chamber. (FIG. 4A &FIG. 4C) Spray freeze dried powder formulations prepared with ultrasonic nozzle (SFD-US) and two-fluid nozzle (SFD-TFN) at different mixing ratios; (FIG. 4B &FIG. 4D) spray dried powder formulations prepared with ultrasonic nozzle (SD-US) and two-fluid nozzle (SD-TFN) at different mixing ratios. Residual fraction (RF) , nasal fraction (NF) , throat fraction (TF) and fine particle fraction (FPF) were expressed as the percentage by mass of tamibarotene with respect to the recovered dose (FIG. 4A &FIG. 4B) . Linear regression of NF and FPF was plotted against fraction of SFD-TFN (FIG. 4C) and SD-TFN (FIG. 4D) in the formulation. Data were presented as mean ± standard deviation (n = 3) .

FIGs. 5A-5B. Aerosol performance of tamibarotene powder formulations dispersed from nasal device at different flow rates. (FIG. 5A) Spray freeze dried powder formulation (SFD-MIX-1) and (FIG. 5B) spray dried powder formulation (SD-MIX-1) were evaluated by Next Generation Impactor (NGI) coupled with 1 L glass expansion chamber and nasal device operated at 15, 28.3 and 40 L/min. Residual fraction (RF) , nasal fraction (NF) , throat fraction (TF) and fine particle fraction (FPF) were expressed as the percentage by mass of tamibarotene with respect to the recovered dose. Data were presented as mean ± standard deviation (n = 3) .

FIGs. 6A-6B. Aerosol performance of tamibarotene powder formulations for pulmonary delivery. (FIG. 6A) Spray freeze dried formulation (SFD-TFN) and (FIG. 6B) spray dried formulations (SD-TFN) , both prepared with two-fluid nozzle, were evaluated by NGI coupled with nasal device, Breezhaler and Handihaler at different flow rates. The glass expansion chamber was only used with the nasal device. Data were presented as mean ± standard deviation (n = 3) .

FIGs. 7A-7B. Dissolution profiles of the emitted fraction (EF) of tamibarotene powder formulations. The EF was collected from a dosage unit sampling apparatus (DUSA) after dispersion from the nasal powder device. The dissolution was performed at 37 ℃ in (FIG. 7A) 10 mL of simulated nasal fluid and (FIG. 7B) 100 mL simulated lung fluid for up to 24 h. Unformulated tamibarotene was used as comparison. Data were presented as mean ± standard deviation (n = 3) .

FIG. 8 Differential scanning calorimetry (DSC) thermograms of tamibarotene powder formulations. The thermograms of spray freeze dried (SFD) and spray dried (SD) powder formulations of tamibarotene. Unformulated tamibarotene, HPBCD and physical mixture of unformulated tamibarotene and HPBCD were used as comparison. Negative peak in DSC thermogram represents endothermic events.

FIG. 9. Aerosol performance of monoclonal antibody powder formulations dispersed from nasal device operated at a flow rate of 28.3 L/min. The formulations were evaluated by Next Generation Impactor (NGI) coupled with 1 L glass expansion chamber. Residual fraction (RF) , nasal fraction (NF) , throat fraction (TF) and fine particle fraction (FPF) were expressed as the percentage by mass of 2-hydroxypropyl-β-cyclodextrin (HPBCD) with respect to the recovered dose. N1 = SD formulations prepared with two-fluid nozzle; N2 = SD formulation prepared with ultrasonic nozzle; N3 = mixed formulations of N1 and N2 at 1: 1 mass ratio; N4 = mixed formulations of N1 and N2 at 3: 7 mass ratio; mixed formulations of N1 and N2 at 7: 3 mass ratio.

FIG. 10. Volumetric particle size distribution of monoclonal antibody powder formulations prepared by spray drying. The powders were dispersed using a nasal powder device and evaluated by laser diffraction. N1 = SD formulations prepared with two-fluid nozzle; N2 = SD formulation prepared with ultrasonic nozzle; N3 = mixed formulations of N1 and N2 at 1: 1 mass ratio; N4 = mixed formulations of N1 and N2 at 3: 7 mass ratio; mixed formulations of N1 and N2 at 7: 3 mass ratio.

FIG. 11. Scanning electron microscopy (SEM) images of monoclonal antibody powder formulations prepared by spray drying (SD) . N1 = SD formulations prepared with two-fluid nozzle; N2 = SD formulation prepared with ultrasonic nozzle; N3 = mixed formulations of N1 and N2 at 1: 1 mass ratio.

DETAILED DISCLOSURE OF THE INVENTION

Selected Definitions

As used herein, the singular forms “a” , “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including” , “includes” , “having” , “has” , “with” , or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising” . The transitional terms/phrases (and any grammatical variations thereof) “comprising” , “comprises” , “comprise” , “consisting essentially of” , “consists essentially of” , “consisting” and “consists” can be used interchangeably.

The phrases “consisting essentially of” or “consists essentially of” indicate that the claim encompasses embodiments containing the specified materials or steps and those that do not materially affect the basic and novel characteristic (s) of the claim.

The term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured, i.e., the limitations of the measurement system. In the context of compositions containing amounts of ingredients where the terms “about” is used, these compositions contain the stated amount of the ingredient with a variation (error range) of 0-10%around the value (X ± 10%) . In other contexts the term “about” is provides a variation (error range) of 0-10%around a given value (X ± 10%) . As is apparent, this variation represents a range that is up to 10%above or below a given value, for example, X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ±9%, or X ± 10%.

In the present disclosure, ranges are stated in shorthand to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. Values having at least two significant digits within a range are envisioned, for example, a range of 5-10 indicates all the values between 5.0 and 10.0 as well as between 5.00 and 10.00 including the terminal values. When ranges are used herein, combinations and subcombinations of ranges (e.g., subranges within the disclosed range) and specific embodiments therein are explicitly included.

As used herein, the term “subject” refers to an animal, needing or desiring delivery of the benefits provided by a drug. The animal may be for example, humans, pigs, horses, goats, cats, mice, rats, dogs, apes, fish, chimpanzees, orangutans, guinea pigs, hamsters, cows, sheep, birds, chickens, as well as any other vertebrate or invertebrate. These benefits can include, but are not limited to, the treatment of a health condition, disease or disorder; prevention of a health condition, disease or disorder; immune health; enhancement of the function of an organ, tissue, or system in the body. The preferred subject in the context of this invention is a human. The subject can be of any age or stage of development, including infant, toddler, adolescent, teenager, adult, or senior.

As used herein, the terms “therapeutically-effective amount, ” “therapeutically-effective dose, ” “effective amount, ” and “effective dose” are used to refer to an amount or dose of a compound or composition that, when administered to a subject, is capable of treating, preventing, or improving a condition, disease, or disorder in a subject. In other words, when administered to a subject, the amount is “therapeutically effective. ” The actual amount will vary depending on a number of factors including, but not limited to, the particular condition, disease, or disorder being treated, prevented, or improved; the severity of the condition; the weight, height, age, and health of the patient; and the route of administration.

As used herein, the term “treatment” refers to eradicating, reducing, ameliorating, or reversing a sign or symptom of a health condition, disease or disorder to any extent, and includes, but does not require, a complete cure of the condition, disease, or disorder. Treating can be curing, improving, or partially ameliorating a disorder. “Treatment” can also include improving or enhancing a condition or characteristic, for example, bringing the function of a particular system in the body to a heightened state of health or homeostasis.

As used herein, “preventing” a health condition, disease, or disorder refers to avoiding, delaying, forestalling, or minimizing the onset of a particular sign or symptom of the condition, disease, or disorder. Prevention can, but is not required, to be absolute or complete; meaning, the sign or symptom may still develop at a later time. Prevention can include reducing the severity of the onset of such a condition, disease, or disorder, and/or inhibiting the progression of the condition, disease, or disorder to a more severe condition, disease, or disorder.

In some embodiments of the invention, the method comprises administration of multiple doses of the compounds of the subject invention. The method may comprise administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or more therapeutically effective doses of a composition comprising the compounds of the subject invention as described herein. In some embodiments, doses are administered over the course of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days, or more than 30 days. The frequency and duration of administration of multiple doses of the compositions is such as prevent or treat a viral infection. Moreover, treatment of a subject with a therapeutically effective amount of the compounds of the invention can include a single treatment or can include a series of treatments. It will also be appreciated that the effective dosage of a compound used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of testing for a virus. In some embodiments of the invention, the method comprises administration of the compounds at several time per day, including but not limiting to 2 times per day, 3 times per day, and 4 times per day.

As used herein, an “isolated” or “purified” compound is substantially free of other compounds. In certain embodiments, purified compounds are at least 60%by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight of the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis.

By “reduces” is meant a negative alteration of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

By “increases” is meant as a positive alteration of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

As used herein, a “pharmaceutical” refers to a compound manufactured for use as a medicinal and/or therapeutic drug.

As used herein, “median aerodynamic diameter” can be defined as the diameter at which 50%of the particles in the aerodynamic size distribution lie above and 50%of the particles in the aerodynamic size distribution lie below that diameter. The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All references cited herein are hereby incorporated by reference in their entirety.

Preparation and Compositions of Bimodal Particle Size Distribution for Use in Treatment of Respiratory Infections

The subject invention pertains to compositions for treating or preventing respiratory infections or a symptom thereof in a subject and methods of synthesizing said compositions.

In certain embodiments, the compositions comprise particles of two distinct sizes, in which the particles contain antiviral compounds. In certain embodiments one group of particles can be at least 10 μm or about 10 μm to about 150 μm in aerodynamic diameter, which can be used for nasal deposition. In certain embodiments, a second group of particles can be about 1 μm to about 5 μm in aerodynamic diameter, which can be used for lung deposition. In certain embodiments, a third group of particles sized between about 5 μm to about 10 μm in aerodynamic diameter can be in the composition, but, preferably, particles sized between about 5 μm to about 10 μm are not present in or are present in quantities significantly less than particles of at least 10 μm in aerodynamic diameter or about 1 μm to about 5 μm in aerodynamic diameter, which can avoid unintended swallowing and minimize systemic exposure.

In certain embodiments, the compositions comprise particles of two distinct sizes, in which the particles contain antiviral compounds. In certain embodiments one group of particles can be at least 10 μm or about 10 μm to about 150 μm in median aerodynamic diameter, which can be used for nasal deposition. In certain embodiments, a second group of particles can be about 1 μm to about 5 μm in median aerodynamic diameter, which can be used for lung deposition. In certain embodiments, a third group of particles sized between about 5 μm to about 10 μm in median aerodynamic diameter can be in the composition, but, preferably, particles sized between about 5 μm to about 10 μm are not present in or are present in quantities significantly less than particles of at least 10 μm in median aerodynamic diameter or about 1 μm to about 5 μm in median aerodynamic diameter, which can avoid unintended swallowing and minimize systemic exposure.

In certain embodiments, the bimodal particle compositions can be created using spray free drying (SFD) and/or spray drying (SD) . In certain embodiments, SFD can be carried out in a two-stage operation –spray freezing followed by freeze drying. The feed solution can be prepared by dissolving a therapeutic agent, such as, for example, tamibarotene, and an excipient, such as, for example, 2-hydroxypropyl-β-cyclodextrin (HPBCD) at a ratio of about 3 to about 7 (drug: excipient) (w/w) , although the ratio of drug to excipient can vary from about 1: 9 to about 9: 1 (drug: excipient) , in about 40%to about 70%or about 55%Tert-Butyl alcohol (TBA) (v/v) to a final solute concentration of about 1%to about 10%or about 4% (w/v) .

In certain embodiments, any therapeutic agents that target the entire respiratory tract from the nose to the lungs can be used. These agents can include antimicrobial agents such as, for example, antivirals, antibiotics, antifungals and antiparasitic drugs for respiratory tract infections, therapeutic drugs such as, for example, mucolytics for acute and chronic respiratory diseases, and therapeutic macromolecules, such as, for example, antibodies and vaccines. Exemplary therapeutic agents can include tamibarotene, remdesivir, clofazimine, ribavirin, oseltamivir, peramivir, zanamivir, baloxavir marboxil, chloroquine, favipiravir, triazavirin, umifenovir, and molnupiravir. In certain embodiments, cyclodextrins (β-Cyclodextrin -BCD, Sulfobutylether-β-cyclodextrin (SBECD) ) and other solubilizing agents other than HPBCD may also be used for insoluble drugs to form an inclusion complex with the therapeutic agent. Other excipients, such as, for example, fillers (e.g. lactose, mannitol) and mucoadhesive agents (e.g., chitosan) , may also be added. TBA is considered to be generally safe for use in the preparation of inhalation formulation (McGregor, 2010) ; although, other organic solvents can be used as an alternative to TBA, including, for example, ethanol.

In certain embodiments, the antiviral compound is an antibody. In certain embodiments, the antibody is a neutralizing antibody, monoclonal antibody, antigen binding fragment (Fab) , single chain variable fragment (scFv) , or other antibody fragment. In certain embodiments, the antibody can be, for example, WKS13, IL4Rα, palivizumab, bamlanivimab (LY-CoV555, Ab169, or LY3819253) , etesevimab (LY-CoV016, CB6, JS016, or LY3832479) , casirivimab (REGN-COV2) , imdevimab (REGN10933 or REGN10987) , tixagevimab, cilgavimab, sotrovimab (VIR-7832 or GSK4182136) , regdanvimab (CT-P59) , bebtelovimab, amubarvimab, romlusevimab, VIR-7831 (S309) , AZD7442 (COV2-2196) , COV2-2130, AZD8895, AZD1061, BRII-196 (1F11) , BRII-198, ADG20 (ADG-2 parent or ADI-55688) , BGB-DXP593 (BD-368-2) , ABBV-47D11 (47D11) , ABBV-2B04 (2B04) , or any combination thereof. In certain embodiments, the antibody can be used to treat a disease of the upper and lower respiratory tract, including, for example, COVID-19, SARS, Middle East Respiratory Syndrome (MERS) , and an influenza inflection. In certain embodiments, the antibody, such as, for example, WKS13, can bind to the Receptor Binding Domain of SARS-CoV-2. The antibody can be at a concentration of about 1%to about 25%, about 5%to about 15%, or about 10%. In certain embodiments, the vaccine is an mRNA vaccine, viral vector vaccine, inactivated vaccine, live-attenuated vaccine, or protein-based vaccine.

In certain embodiments, the subject composition can be used to deliver antibodies, pharmaceutical drugs, or nucleotides (e.g., DNA or RNA) that target respiratory viral infections that affect both upper and lower airways, such as, for example, COVID-19, influenza, SARS, Mycobacterium tuberculosis, Streptococcus pneumoniae, respiratory syncytial virus (RSV) , and MERS. In certain embodiments, the subject composition and methods thereof can be used to deliver vaccines against any respiratory infections, such as, for example, COVID-19, influenza, tuberculosis, respiratory syncytial virus infection or pneumococcal infections.

In certain embodiments, the composition can further comprise a stabilizer and/or dispersion enhanced. In preferred embodiments, the stabilizer/dispersion enhancer is an amino acid, including, for example, leucine, isoleucine, phenylalanine, alanine, glycine, tryptophan, and valine. The amino acid can be at a concentration of about 1%to about 50%, about 10%to about 30%, or about 20%.

The solution of the therapeutic agent, excipient, and solvent can be maintained at about 30 ℃ to about 40 ℃ or about 37 ℃ before being loaded into a syringe that can be connected to a syringe pump. The liquid feed rate can be controlled at about 1 mL/min to about 2.5 mL/min or about 1.5 mL/min. About half of the feed solution can be atomized using a two-fluid nozzle (TFN) operated at a nitrogen gas flow rate of about 473 L/h (40 mm height rotameter) to about 742 L/h (60 mm height rotameter) or about 601 L/h. The ratio of feed solutions can be varied depending on how much of each particle size is required for the final mixed formulation. If a 1: 1 mix is required, then the feed solution can be divided into half to obtain an equal amount of each particle size distribution. If other mixing ratios are desired, then the feed solution can be divided accordingly. In certain embodiments, the two-fluid nozzle can have an internal diameter of internal diameter of 0.7 mm. The other half, or other amount depending on the final mixing ratio desired, of the feed solution can be atomized using an ultrasonic nozzle operated at about 20 kHz to about 200 kHz or about 130 kHz powered by a digital ultrasonic generator. The sprayed droplets can be instantly frozen in stainless-steel collectors containing liquid nitrogen and can then transferred into a pre-cooled freeze dryer. The freeze dryer can be operated at a vacuum pressure below 0.14 mBar or between about 0.133 mBar to about 0.014 mBar and programmed to maintain a primary drying temperature of about -10 ℃ to about -50 ℃ or about -25 ℃ for about 10 h to about 60 h or about 40 h, optionally, followed by gradually increasing to a secondary drying temperature of about 20 ℃ to about 40 ℃ or about 20 ℃ for about 1 second to about 12 hours, about 1 hour to about 8 hours, or about 4 hours, depending on the residual moisture level, with a higher moisture level requiring a longer drying time, and can be stabilized at room temperature. In certain embodiments, the powder can be stabilized for about 20 h. The SFD powders produced by the two different nozzles can be collected separately. The production yield can be calculated as the percentage of total mass of powder collected to the initial solute mass in the feed solution. The powder can be stored in a desiccator at room temperature.

In certain embodiments, spray drying can be performed using a spray dryer with a high-performance cyclone in suction mode and closed loop configuration, with an inlet temperature of about 100 ℃ to about 150 ℃ or about 150 ℃ and aspiration rate at about 100% (approximately 35 m³/h) . The feed solution for spray drying can be prepared by dissolving a therapeutic agent, such as, for example, tamibarotene or other agents as previously described, and an excipient, such as, for example, 2-hydroxypropyl-β -cyclodextrin (HPBCD) or other excipients as previously described, at a ratio of about 3 to about 7 (drug: excipient) (w/w) , although the ratio of drug to excipient can vary from about 1: 9 to about 9: 1 (drug: excipient) in about 10%to about 90%or about 70%ethanol (v/v) to a final solute concentration of about 1%to about 10%w/w or about 2%w/v. Ethanol is considered to be generally safe for use in the preparation of inhalation formulation (McGregor, 2010) ; although, other organic solvents can be used as an alternative to TBA, including, for example, DMSO or methanol. About half of the feed solution can be sprayed at a rate of about 0.3 mL/min to about 30 mL/min or about 1.2 mL/min with a TFN controlled at a nitrogen gas flow rate of about 473 L/h (40 mm height rotameter) to about 742 L/h (60 mm height rotameter) or about 601 L/h. The ratio of feed solutions can be varied depending on how much of each particle size is required for the final mixed formulation. If a 1: 1 mix is required, then the feed solution can be divided into half to obtain an equal amount of each particle size distribution. If other mixing ratios are desired, then the feed solution can be divided accordingly. In certain embodiments, the two-fluid nozzle can have an internal diameter of internal diameter of 0.7 mm. In certain embodiments, about the other half, or other amount depending on the final mixing ratio desired, of the feed solution can be sprayed at a rate of about 1 mL/min to about 9 mL/min or about 2.4 mL/min with an ultrasonic nozzle operated at 0.1 W to about 15W, 1 W to about 15 W, or about 0.9 W. The SD powders produced by the two different nozzles can be collected separately. The production yield can be calculated as the percentage of total mass of powder collected to the initial solute mass in the feed solution. The powder can be stored in a desiccator at room temperature.

The dry powder formulations prepared with the same drying method but different atomization nozzle can be mixed at different mass ratios by shaking by hand or by using, for example, rotating blenders, rotating drums, V-mixer, double-cone blender, tote blender or, preferably, shaker-mixer type T2F (Willy A. Bachofen AG Maschinenfabrik, Basel, Switzerland) . A total of about 10 mg to about 4 kg, about 10 mg to about 1 kg, about 10 mg to about 100 mg, or about 40 mg of powder was placed into a glass vial and subjected to a constant rotational speed of about 23 rpm to about 69 rpm or about 49 rpm. The mixing can be carried out at room temperature and the samples can be subjected to translational and rotational mixing for about 5 min to about 30 min or about 10 min.

In certain embodiments, the mixer can apply the powder blending principles of rotation, translation and inversion (Sommier et al., 2001) . The powders can be mixed within the cylindrical mixing chamber with an alternating and rhythmic motion to achieve blending.

Treatment of Respiratory Infections with Bimodal particles

Administration of the bimodal particles can be carried out in the form of a powder formulation containing a therapeutically effective amount of the active ingredient (antiviral agent) . Administration of the bimodal particles can be intranasally.

In some embodiments, an amount of the bimodal particles can be administered 1, 2, 3, 4, or times per day, for 1, 2, 3, 4, 5, 6, 7, or more days. Treatment can continue as needed, e.g., for several weeks. Optionally, the treatment regimen can include a loading dose, with one or more daily maintenance doses.

In certain embodiments, the particles can be administered after a viral infection. The particles can limit or prevent complications or symptoms of the previous viral infection.

Another aspect of the invention concerns a method for inhibiting a human coronavirus infection in a human cell, comprising contacting a viral particle and/or infected cell with particle compositions of the subject invention, before or after the viral particle infects a cell.

The human coronavirus may be any type or subgroup, including alpha, beta, gamma, and delta. In some embodiments of the aforementioned methods of the invention, the human coronavirus is selected from among SARS-CoV-2, SARS-CoV, and MERS-CoV. In some embodiments of the aforementioned methods of the invention, the human coronavirus is a common human coronavirus, such as type 229E, NL63, OC43, and HKU1.

Another aspect of the invention concerns a method for inhibiting an influenza virus infection in a human cell, comprising contacting a viral particle and/or infected cell with particle compositions of the subject invention, before or after the viral particle infects a cell.

The influence may be any type or subgroup, including influenza A, influenza B, and influenza C, and mutants thereof. Influenza viruses contemplated herein include those viruses that have two antigenic glycosylated enzymes on their surface: neuraminidase and hemagglutinin. Various subtypes of influenza virus that can be treated using the materials and methods of the invention include, but are not limited to, the HINI, H1N2, H2N2, H3N2, H3N8, H5N1, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, and H10N7 subtypes including the following subtypes commonly known as the "Spanish Flu, " "Asian Flu, " "Hong Kong Flu, " "Avian Flu, " "Swine Flu, " "Horse Flu, " and "Dog Flu. "

For the treatment of respiratory virus infections, the nasal cavity and the deep lung region are the two specific sites that the dual targeting formulations of a therapeutic agent can be targeted. In general, the aerosol performance of a good dual targeting powder formulation should have a NF (for nasal deposition) and FPF (for lung deposition) in an appropriate ratio. As SARS-CoV-2 and other respiratory viruses affect the upper and lower respiratory tract with increasing infectivity, it is crucial that high doses of the antiviral drug can reach both infected sites to achieve optimal therapeutic effect. In certain embodiments, mixing in a 1: 1 ratio of the bimodal powder can enable efficient powder deposition in both the upper and lower airways. The fraction of particles deposited in the throat can be minimal and there can be two major fractions of the formulation that deposit separately in the nasal (NF) and the lung region (FPF) .

In certain embodiments, the ratios of the particles targeting the NF and FPF can vary. The ratio of NF: FPF can vary linearly with the mixing ratio. The ratio can depend on the individual formulations before the mixing process. For an optimized nasal and pulmonary formulation, the range in principle can be from about 100: 0 to about 0: 100. For the SFD tamibarotene bimodal particles, the range can vary from 87: 8 to 57: 33. For the SD tamibarotene bimodal particles, the range can vary from 94: 2 to 49: 44. In certain embodiments, the mixing ratio may be manipulated to tailor the ratio of NF: FPF in the mixed formulation for targeted aerosol deposition in the nasal cavity and the lung depending type of respiratory infection, stage of infection, and/or physical characteristics of the infected subject. By varying the ratio deposited at different sites, the formulation can target a specific site, either the upper or lower respiratory tract, to achieve a higher drug concentration in the nose or lung. For example, for upper respiratory tract infections, the formulation can have a higher NF: FPF ratio. Conversely, for lower respiratory tract infections, the formulation can have a lower NF: FPF ratio. The stage of infection may also influence the primary infected cells. At an early stage of infection, the nose may be the initial primary infected cells before further viral spread throughout the respiratory tract. Hence, a higher NF: FPF ratio may allow more drugs to be delivered to the nasal cavity. At a later stage of infection, we may want an NF:FPF ratio closer to 50: 50 to allow uniform distribution of drug throughout the respiratory tract to allow maximum targeting of all infected cells. Infected patient with symptomatic physical characteristics, such as increased mucus secretion in the nose or chronic conditions of the lung, may also benefit from varied NF: FPF ratio improve drug delivery to specific sites.

In certain embodiments, the dissolution profile of a therapeutic agent, such as, for example, tamibarotene can be enhanced, as the subject methods improve solubility by producing amorphous solid dispersions of the therapeutic agent. In certain embodiments, tamibarotene can be incorporated into the hydrophobic internal cavity of HPBCD. In certain embodiments, the subject compositions can be in an amorphous state, which can enhance the dissolution rate as compared to the crystalline unformulated therapeutic agents. In certain embodiments, the spherical and porous structure of SFD particles and the small particle size with large surface area of SD particles for hydration can lead to faster dissolution.

MATERIALS AND METHODS

Materials

Tamibarotene was purchased from Dalian Meilun Biotechnology Co., Ltd (China) . 2-hydroxypropyl-β -cyclodextrin (HPBCD) was purchased from Sigma-Aldrich (Saint Louis, USA) . Tert-Butyl alcohol (TBA) was obtained from Meryer Chemical Technology (Shanghai, China) . Ethanol was obtained from VWR BDH Chemicals (VWR International S. A. S., Fontenay-sous-Bois, France) . Methanol and acetonitrile (HPLC grade) were purchased from Anaqua Chemicals Supply (Cleveland, USA) . Acetic acid (HPLC grade) was purchased from Fisher Scientific (Loughborough, UK) . All solvents and reagents were of analytical grade.

Preparation of dry powder formulation by spray freeze drying (SFD)

Spray freeze drying (SFD) was carried out in a two-stage operation –spray freezing followed by freeze drying. The feed solution was prepared by dissolving tamibarotene and HPBCD at 3: 7 ratio (w/w) in 55%TBA (v/v) to a final solute concentration of 4% (w/v) (Table 1) . To prevent freezing of TBA (freezing point: 25.4 ℃) , the solution was maintained at 37 ℃ before being loaded into a pre-warmed 10 mL syringe (Philippines) that was connected to a syringe pump (210 Syringe Pump, KD Scientific, MA, USA) . The liquid feed rate was controlled at 1.5 mL/min. Half of the feed solution was atomized using a two-fluid nozzle (TFN; Büchi with an internal diameter of 0.7 mm, Switzerland) operated at a nitrogen gas flow rate of 601 L/h. The other half of the feed solution was atomized using an ultrasonic nozzle (US; 130K50ST, Farmingdale, NY, USA) operated at 130 kHz powered by a digital ultrasonic generatorNY, USA) . The sprayed droplets were instantly frozen in stainless-steel collectors containing liquid nitrogen and were then transferred into a pre-cooled freeze dryer (6 Litre Benchtop Freeze Dry System with Stoppering Tray Dryer, Labconco Corporation, Missouri, USA) . The freeze dryer was operated at a vacuum pressure below 0.14 mBar and programmed to maintain a primary drying temperature of -25℃ for 40 h, followed by gradually increasing to a secondary drying temperature of 20 ℃ in 4 h, and finally stabilised at room temperature for at least 20 h. The SFD powders produced by the two different nozzles were collected separately. The production yield was calculated as the percentage of total mass of powder collected to the initial solute mass in the feed solution. The powder was stored in a desiccator at room temperature before analysis.

Preparation of dry powder formulation by spray drying (SD)

Spray drying was performed using a laboratory scale spray dryer with a high-performance cyclone in suction mode and closed loop configuration (Mini Spray Dryer B-290, Dehumidifier B-296 and Inert Loop B-295; Büchi Labortechnik, Flawil, Switzerland) , with an inlet temperature of 150 ℃ and aspiration rate at 100% (approximately 35 m³/h) . The feed solution for spray drying was prepared by dissolving tamibarotene and HPBCD at 3: 7 ratio (w/w) in 70%ethanol (v/v) to a final solute concentration of 2%w/v (Table 1) . Half of the feed solution was sprayed at a rate of 1.2 mL/min with a two-fluid nozzle (TFN; Büchi with an internal diameter of 0.7 mm, Switzerland) controlled at a nitrogen gas flow rate of 601 L/h. The other half of the feed solution was sprayed at a rate of 2.4 mL/min with an ultrasonic nozzle (US; Büchi for particle size 10 to 60 μm, Switzerland) operated at 0.9 W. The SD powders produced by the two different nozzles were collected separately. The production yield was calculated as the percentage of total mass of powder collected to the initial solute mass in the feed solution. The powder was stored in a desiccator at room temperature before analysis.

Powder mixing to produce dual particle size formulation

The dry powder formulations prepared with the same drying method but different atomization nozzle were mixed at different mass ratios using ashaker-mixer type T2F (Willy A. Bachofen AG Maschinenfabrik, Basel, Switzerland) (Table 1) . A total of 40 mg of powder was placed into a 50 mL glass vial and subjected to a constant rotational speed of 49 rpm. The mixing was carried out at room temperature and the samples were subjected to translational and rotational mixing for 10 min.

Table 1. Formulations of tamibarotene dry powder produced by spray freeze drying (SFD) and spray drying (SD) . *w/w ratio was used in the mixed formulation.

Morphology study using scanning electron microscopy (SEM)

The morphology of tamibarotene powder formulations were observed under the field emission SEM (Hitachi S-4800 N, Tokyo, Japan) at 5.0 kV. The powders were sprinkled onto carbon double-sided tape and mounted onto SEM stubs. Excess layers of powder were removed by tapping the stubs and blowing with clean compressed air. To avoid charging interferences during SEM, the stubs with samples were sputter-coated with approximately 13 nm of gold-palladium alloy in 90 s using a sputter coater (Q150T PLUS Turbomolecular Pumped Coater, Quorum, UK) .

Drug quantification using high performance liquid chromatography (HPLC)

The amount of tamibarotene in each formulation was quantified using HPLC with photodiode array detector (Agilent 1260 Infinity; Santa Clara, USA) . The dry powders were weighed and dissolved in methanol to a final volume of 25 mL. The dissolved samples were filtered through a 0.45-μm nylon membrane filter and quantified by HPLC. A sample of 25 μL was injected into a C-18 column (Agilent Prep –C18, 4.6 mm × 250 mm, 5 μm) with a mobile phase composed of acetonitrile and 5%acetic acid (80 : 20, v/v) , running at a flow rate of 1 mL/min. Tamibarotene was detected at a wavelength of 280 nm with a retention time of 6.2 min. The measurements of drug content in each formulation were performed in triplicate. Drug content was calculated as the percentage of tamibarotene detected in the formulation to the total mass of powder.

Thermogravimetric Analysis (TGA)

The residual moisture of the tamibarotene powders were determined using thermogravimetric analysis (TGA, TA Instruments, Newcastle, DE, USA) . Approximately 3 to 5 mg of powder sample was loaded onto a titanium pan and kept at 25 ℃ until mass equilibrium was reached. The sample was heated to 120 ℃ at a constant rate of 10 ℃/min. The amount of residual moisture was calculated as the change in powder mass upon heating.

Particle size distribution by laser diffraction

The volumetric size distribution of the dry powders was measured using a HELOS/KR laser diffractometer coupled with an Inhaler module (Sympatec, Germany) . To determine the particle size of the dry powder aerosols when dispersed, a nasal powder device (Unit Dose System Powder Nasal Spray, Aptar Pharma, France) was filled with 2.0 ± 0.5 mg of powder and mounted at a 30° angle on the central unit of the Inhaler module. All measurements were conducted with 100 mm (R3) lens (measuring range 0.45 -175 μm) with appropriate trigger and stop conditions. Particle size distribution was calculated using WINDOX 5 based on the enhanced Fraunhofer theory. The particle size data were expressed as D₁₀, D₅₀, and D₉₀, which represent the equivalent spherical volume diameters at 10%, 50%and 90%cumulative volumes, respectively. Span was calculated as (D₉₀-D₁₀) /D₅₀. For each formulation, the most representative volumetric particle size distribution data were plotted on a graph for comparison.

Aerosol performance by cascade impaction

The aerodynamic size distribution of the dry formulations was evaluated using a Next Generation Impactor (NGI, Copley, Nottingham, UK) coupled with a 1 L expansion chamber (Copley, Nottingham, UK) operated at 28.3 L/min. Before dispersion, a thin layer of silicon grease (LPS Laboratories, Illinois, GA, USA) was coated onto the stages of the cascade impactors to reduce particle bounce and wall losses. A powder mass of 2.0 ± 0.1 mg was loaded into a nasal powder device and dispersed at a 30° angle into the 1 cm inlet hole of the expansion chamber. After dispersion, methanol was used to rinse and dissolve all powder deposited on the stages of the NGI and expansion chamber. The collected samples were filtered through a 0.45-μm membrane filter and analysed by HPLC. For every powder formulation, dispersions were performed in triplicates. The recovered dose was defined as the total mass of tamibarotene quantified on all stages of the cascade impactor in a single run. Residual fraction (RF) referred to the fraction of powder that was undispersed and remained in the dispersion device. Nasal fraction (NF) was defined as the percentage fraction of particles that exited the dispersion device and had an aerodynamic diameter of more than 10.0 μm. Throat fraction (TF) was defined as the percentage of particles with aerodynamic diameter between 5.0 μm and 10.0 μm. Fine particle fraction (FPF) was defined as the percentage fraction of particles with aerodynamic diameter less than 5.0 μm. All fractions (RF, NF, TF and FPF) were calculated with respect to the recovered dose.

Dissolution study

Dissolution study was performed with the emitted fraction (EF) of the powder formulation, which is defined as the fraction of powder that exited the dispersion device. Modelling the nasal environment, a 100 mL jacketed beaker containing 10 mL of simulated nasal fluid (SNF) was used to examine the dissolution rate of the tamibarotene formulations prepared with ultrasonic nozzle (SFD-US and SD-US) . Simulated nasal fluid was prepared with 7.45 mg/mL of sodium chloride, 1.29 mg/mL of potassium chloride and 0.32 mg/mL of calcium chloride (Trenkel and Scherliess, 2021) . The pH of resulting solution was adjusted to 6.5 with 0.1 M HCl. The temperature was maintained at 37 ℃ and the medium was stirred at 75 rpm with a magnetic bar. The EF of the nasal formulations were collected by a dosage unit sampling apparatus (DUSA, Copley Scientific, UK) after dispersion from the nasal powder device with a flow rate and dispersion duration of 28.3 L/min and 4 s respectively. A powder mass of 2.0 ± 0.1 mg was dispersed, and the EF was collected on a glass fibre filter paper which was then transferred into the jacketed beaker. At pre- determined time intervals, 0.5 mL of dissolution medium was withdrawn and filtered through a 0.45-μm membrane filter before quantification by HPLC. After each sampling point, the medium was made up to 10 mL by replacing with an equal volume of pre-warm fresh dissolution medium. To simulate the lung environment, a 500 mL jacketed beaker contained 100 mL of simulated lung fluid (SLF) as the dissolution medium (Liao et al., 2021) . The temperature was maintained at 37 ℃ and the medium was stirred at 75 rpm with a magnetic bar. The EF of formulations prepared with the two-fluid nozzle (SFD-TFN and SD-TFN) were collected as described above and transferred to the jacketed beaker. At pre-determined time intervals, 1 mL of dissolution medium was withdrawn and filtered through 0.45 μm membrane filter before quantification by HPLC. After each sampling point, the medium was made up to 10 mL by replacing with an equal volume of pre-warm fresh dissolution medium. For comparison, the dissolution profile of equivalent amount of raw tamibarotene powder was studied in the simulated nasal and lung environment without dispersion. All the dissolution tests were carried out in triplicates.

Differential scanning calorimetry

Differential scanning calorimetry (DSC) (DSC 250, TA Instruments, Newcastle, DE, USA) was used to investigate the thermal response of raw tamibarotene, the SFD and SD formulations and their physical mixtures. Approximately 1 mg of powder was weighed, loaded into hermetically sealed aluminium pans and heated from 50 ℃ to 280 ℃ at a constant rate of 10 ℃/min. The thermogram of each sample was obtained.

Statistical Analysis

Statistical analyses were conducted using GraphPad Prism 8.0 for Student's t-test and ANOVA when comparing two or three different conditions, respectively. A significance level of α = 0.05 was used throughout the study.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1-DRUG CONTECT, PRODUCTION YIELD AND RESIDUAL MOISTURE

The amount of tamibarotene present in the powder formulations was measured by HPLC (Table 2) . In all the tested formulations, the measured drug content was close to the theoretical value of 30 %w/w of tamibarotene. For both SFD and SD techniques, the production yield varied with the nozzle used for atomization. The ultrasonic nozzle and SFD had a higher yield compared to the two-fluid nozzle and SD, respectively (Table 2) . All powder formulations demonstrated low levels of residual moisture (< 6 %w/w) , although SD powder generally had a lower moisture content than its SFD counterpart.

Table 2. Production yield, tamibarotene content and residual moisture of the spray freeze drying (SFD) and spray drying (SD) tamibarotene formulations. Drug content data are presented as mean (standard deviation) , n = 3.

EXAMPLE 2-PARTICLE SIZE DISTRIBUTION

The volumetric particle size distribution of tamibarotene powder formulations was measured by laser diffraction after the powder was dispersed from a nasal powder device. The data were presented as incremental size distribution (FIGs 1A-1B) , D₁₀ D₅₀ D₉₀ and span (Table 3) . Comparing the formulations prepared with the same nozzle, particles produced by SFD were generally larger than their SD counterparts. For both SFD and SD formulations, particles produced using the ultrasonic nozzle had a larger D₅₀ as compared to those produced by the two-fluid nozzle. At 1: 1 mixing ratio, the D₅₀ of the mixed formulations (SFD-MIX-1 and SD-MIX-1) tended to be closer to the D₅₀ of the smaller particle size formulation prepared with two-fluid nozzle (SFD-TFN and SD-TFN, respectively) . The mixed SD and SFD formulations were examined at several mixing ratios. A bimodal size distribution was observed in SD-MIX-0.25, SD-MIX-1 and SD-MIX-2 formulations (at 3: 1, 1: 1 and 1: 2 of SD-US to SD-TFN ratio, respectively) , with each of the two separate peaks corresponding to the individual peaks of SD-US and SD-TFN. When the proportion of SD-TFN increased in the mixed formulation, the peak corresponding to the smaller particle size increased while the peak corresponding to the larger particle size diminished. As the content of SD-TFN further increased (SD-MIX-3 and SD-MIX-7 at 1: 3 and 1: 7 ratio, respectively) , a unimodal distribution was observed with the mode similar to that of the SD-TFN formulation. In contrast, the SFD mixed formulations tended to have unimodal distributions with broader peaks due to the wider distribution of particle size produced by SFD. When the proportion of SFD-TFN increased (SFD-MIX-2, SFD-MIX-3, and SFD-MIX-7 at 1: 2, 1: 3 and 1: 7 ratio, respectively) , the magnitude of peak increased and shifted to the left, indicating a larger proportion of smaller particles. SFD-MIX-0.25 formulation (3: 1 of SFD-US to SFD-TFN ratio) demonstrated a very broad distribution implying a relatively even distribution of particle sizes.

Table 3. Volumetric particle size distribution of tamibarotene powder formulations. The powders were dispersed using the nasal powder device and the particle size distribution was measured by laser diffraction. Data are presented as mean (standard deviation) , n = 3.

EXAMPLE 3-MORPHOLOGY

The morphology of the tamibarotene powder formulations were visualized by scanning electron microscopy (SEM) under × 2,500 magnification to examine the particle surface properties (FIG. 2) and under × 500 magnification to examine the overall particle distribution (FIG. 3) . While unformulated tamibarotene exists as irregular rod-shaped crystalline structures, the SFD and SD powders were spherical. The surface of the SFD particles was slightly porous with visible cavities (at high magnification) as compared to the smooth exterior texture of the SD particles. The single formulations exhibited a relatively uniform size distribution. For both SFD and SD processes, the particles produced using the two-fluid nozzle were small (< 5 μm) and seemed to aggregate as compared to the larger discrete particles produced by the ultrasonic nozzle (> 10 μm) . When two single formulations prepared with the same drying method but different nozzle were mixed at a 1: 1 ratio (SFD-MIX-1 and SD-MIX-1) , an amalgam of small and large particles can be clearly observed, displaying two distinct particle sizes. Most of the particles retained their spherical structures after mixing, with some of the smaller particles loosely attached to the surface of the larger particles. Some small fragments can also be seen deposited on the surface of the particles after mixing, further broadening the dual particle size distribution in the mixed formulation. The mixed formulations with different mixing ratio showed distinctly different distributions of particles. When high proportion of SFD-US or SD-US was present in the mixed formulation (SFD-MIX-0.25, SD-MIX-0.25) , large particles (> 10 μm) were the dominant species, with smaller particles on the surface and in the surrounding. As the proportion of SFD-TFN or SD-TFN increased, there were fewer large particles and increasingly more smaller particles (< 10 μm) , as clearly shown in SFD-MIX-7 and SD-MIX-7.

EXAMPLE 4-AEROSOL PERFORMANCE

Suitable range of aerodynamic diameter of the dispersed particles is required for efficient deposition to the appropriate sites along the respiratory tract. The aerosol performance of the tamibarotene powder formulations is presented as RF, NF, TF and FPF which were obtained from the NGI experiments (FIGs. 4A-4D) . All formulations demonstrated excellent dispersion from the nasal device (RF < 10%) . The formulations produced with the ultrasonic nozzle, SFD-US and SD-US exhibited high NF (both over 80%) and low FPF (both below 10%) , suggesting high nasal deposition and low lung deposition. In comparison, aerosol performance of formulations prepared with the two-fluid nozzle exhibited moderate FPF, with 34%for SFD-TFN and 44%for SD-TFN when dispersed with a nasal powder device, indicating the feasibility for lung deposition through intranasal administration.

The rationale of the mixed formulations was to allow the manipulation of NF and FPF by modifying the ratio between US and TFN formulations. Regardless of the mixing ratio, all mixed formulations showed similarly low RF and TF, demonstrating that the mixed dry powder formulations were dispersed and aerosolized effectively from the nasal powder device, with minimal powder deposition at unintended sites. When mixed at a 1: 1 ratio, the NF and FPF values of both SFD-MIX-1 and SD-MIX-1 were close to the average of the individual formulations, achieving a NF to FPF ratio of approximately 71 : 23 and 70 : 25, respectively. When a wider range of mixing ratio was investigated, a linear trend for the ratio of NF to FPF was observed. As the percentage of SFD-TFN or SD-TFN increased in the mixed formulation, FPF increased proportionally until the maximum FPF (33%and 38%, respectively) was reached, while the NF decreased to the minimum value (57%and 49%, respectively) . The NF to FPF ratio for the SD-MIX formulations exhibited a wider range from 85 : 11 (SD-MIX-0.25) to 55 : 38 (SD-MIX-7) as compared to the SFD-MIX formulations with a range from 78 : 17 (SFD-MIX-0.25) to 59 : 32 (SFD-MIX-7) .

To further investigate the effect of flow rate on powder deposition, the aerosol performance of two mixed formulations SFD-MIX-1 and SD-MIX-1 (FIGs. 5A and 5B) were examined at three different flow rates (15, 28.3 and 40 L/min) with powders dispersed from a nasal device. The NF and FPF were compared using one-way ANOVA (Table 4) . For both formulations, the aerosol performance at different flow rates were significantly different (p < 0.05) . While RF and TF remains the same, NF decreased at a higher flow rate, with a corresponding increase in FPF. In our previous study, SFD powder of tamibarotene prepared with the two-fluid nozzle was reported to be suitable for targeting the respiratory tract when aerosolized with the Breezhaler (Liao et al., 2021) . In comparison with the nasal device, SFD-TFN dispersed by the Breezhaler (for oral inhalation) exhibited significantly decreased RF and significantly increased FPF (p < 0.05) (FIGs. 6A and 6B) . There was no significant change in aerosol performance of SFD-TFN when Breezhaler was operated at 60 and 90 L/min. In contrast, the FPF of SD-TFN was not significantly different regardless of the dispersion device and flow rate used for dispersion (Table 4) . However, the RF of SD-TFN significantly increased when dispersed from the Breezhaler, implying inefficient dispersion. Using the Handihaler with a higher resistance and different mechanism of dispersion improved powder dispersion, showing a reduction in RF.

Table 4. One-way ANOVA and Tukey’s multiple comparisons test based on nasal fraction (NF) and fine particle fraction (FPF) . Data were obtained from NGI experiments with nasal spray, Breezhaler and Handihaler operated at different flow rates. *p < 0.05, ns: not significant.

EXAMPLE 5-DISSOLUTION PROFILE

Dissolution study was performed using the EF of the tamibarotene powder formulations. The powders were dispersed using the nasal powder device and collected from the DUSA. Formulations prepared with ultrasonic nozzle (SFD-US and SD-US) were designed for nasal deposition and were evaluated in SNF (FIG. 7A) , while formulations prepared with two-fluid nozzle (SFD-TFN and SD-TFN) were designed for pulmonary deposition and were evaluated in SLF (FIG. 7B) for their dissolution profile. All the tamibarotene powder formulations demonstrated burst-release profiles with a faster dissolution rate compared to unformulated tamibarotene. For formulations prepared with ultrasonic nozzle (for nasal deposition) , more than 50%of tamibarotene in SD-US was dissolved in 1 h while just over 30%of the drug in SFD-US was dissolved in 2 h. The cumulative concentration of both formulations stabilized to around 40%after 4 h. At the end of the experiment (24 h) , a significantly higher amount of tamibarotene (40%) was released from SD-US compared to the unformulated tamibarotene (Student's t-test, p < 0.01) . In comparison, the unformulated tamibarotene exhibited a slower rate of dissolution until the end of the experiment. For formulations prepared with two-fluid nozzle (for pulmonary deposition) , more than 50%of tamibarotene in SD-TFN formulation was dissolved within the first 15 min. In contrast, SFD-TFN dissolved at a faster rate, reaching a maximum of more than 60%of tamibarotene released within the first 5 min. After reaching the peak concentration, there was a slight decrease in the drug concentration of both formulations. The cumulative concentration remained relatively constant for the next 6 h, with about 60%of drug dissolved at the end of the experiment (24 h) . In SLF, the unformulated tamibarotene dissolved at a slower rate with significantly lower amount of drug dissolved (30%) compared to SFD-TFN after 24h (Student's t-test, p < 0.01) .

EXAMPLE 6-POWDER CRYSTALLINITY

The crystallinity of raw drug, excipient and the tamibarotene powder formulations were examined using the DSC (FIG. 8) . In the DSC thermogram of raw unformulated tamibarotene, the two endothermic peaks suggest an initial change in crystalline state at 184 ℃ and a subsequent breakdown of the re-crystallised form at 231 ℃, which corresponds to the melting point of tamibarotene. The excipient HPBCD was amorphous as no significant thermal event was detected. Upon physical mixture of raw tamibarotene with HPBCD, the endothermic peaks can still be detected at the same temperatures (184 ℃ and 231 ℃) . This suggests that raw tamibarotene retained its crystalline state in a physical mixture with HPBCD. After SFD and SD, the formulations became amorphous as shown in their respective thermograms with no significant thermal events.

EXAMPLE 7-PREPARATION OF FEED SOLUTION

The feed solution for spray-drying was prepared by mixing a monoclonal antibody (mAb) with a solution of 2-hydroxypropyl-beta-cyclodextrin (CD; Sigma-Aldrich, USA) in ultrapure water (Barnstead, USA) . The CD was weighed (804.29 mg) and dissolved in a 25-mL volumetric flask. 42 mg of mAb, measured by Bradford protein assay using bovine γ-globulin as standard, was added to 798 mg of CD (24.8 mL) and the solution was made up to 42 mL with ultrapure water. This resulted in a 2%w/v feed solution comprising of 95%w/w CD and 5%w/w mAb. 40 mL was spray-dried and 2 mL was kept as physical mixture.

EXAMPLE 8-SPRAY-DRYING OF mAb FORMULATION

The spray dried powders were prepared with a mini spray dryer (B-290, Switzerland) coupled separately to two types of nozzle: two-fluid nozzle (TFN) and ultrasonic nozzle (USN) . For the TFN, the peristaltic pump rate was 3% (～0.9 mL/min feed flow) and the spray gas (nitrogen) flow was 742 L/hr. For the USN, the pump rate was 8% (～2.4 mL/min feed flow) and the spray gas flow, supplied to maintain the nozzle temperature below 100 ℃ at a pre-determined ultrasonic atomisation power of 1.0 W, was 192 L/hr. For both nozzles, the inlet temperature set was 100 ℃ and the aspiration was 100% (～35 m³/hr) . 20 mL of feed solution was spray-dried using each nozzle. The spray-dried powders, N1 (prepared with TFN) and N2 (prepared with USN) were transferred into glass vials and stored in an auto dry box at room temperature.

Exemplary formulations were created using the WKS13 antibody (Tables 5, 6, 7, 8, 9, and 10) .

Table 5. WKS13 antibody formulations

Table 6. Blank powder formulations containing only excipients without WKS13 mAb produced by spray drying.

Table 7. Volumetric particle size distribution of powder formulations. The powders were dispersed using the nasal powder device and the particle size distribution was measured by laser diffraction. Data are presented as mean (standard deviation) , n = 3.

Table 8. Aerosol performance of powder formulations. The powders were dispersed using the Osmohale inhaler device and evaluated using the Next Generation Impactor (NGI) . Data are presented as mean (standard deviation) , n = 3.

EF: emitted fraction; FPF: fine particle fraction; GSD: geometric standard deviation; MMAD: mass median aerodynamic diameter

Table 9. Dry powder formulations of WKS13 mAb produced by spray drying. The mixed formulations were prepared by blending two single formulations at a specific ratio (w/w) .

Table 10. The production yield, measured mAb contents and residual moisture content of the spray dried powder formulations of WKS13. Data were presented as mean ± standard deviation (n = 3) .

EXAMPLE 9-PREPARATION OF DUAL-TARGET POWDER FORMULATION

An equivalent amount of N1 and N2 (50 mg each) was placed in a glass vial and subjected to high-efficiency mixing using a 3D shaker mixer (WAB, Switzerland) for 10 minutes. The resultant mixture is labelled N3. The aerosol performance (FIG. 9) , particle size distribution (FIG. 10) and morphology (FIG. 11) of the antibody powder formulations were evaluated.

Table 11: Spray drying Output

*Processing yield = percentage of mass of powder in the product collection vessel to mass of total solid loading in feed solution

Table 12. Aerosol performance NGI-HPLC (n=3)

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

EMBODIMENTS

Embodiment 1. A method for prophylactic or responsive treatment of a respiratory infection in a subject or disease of the respiratory tract of a subject or a symptom thereof, said method comprising administering a composition comprising an effective amount of a first particle with a median aerodynamic diameter of at least about 10 μm and a second particle with a median aerodynamic diameter of about 1 μm to about 5 μm, wherein the first particle and the second particle comprise a therapeutic agent.

Embodiment 2. The method of embodiment 1, wherein the respiratory infection is a viral infection.

Embodiment 3. The method of embodiment 2, wherein the viral infection is a coronavirus infection or an influenza infection.

Embodiment 4. The method of embodiment 3, wherein the coronavirus is SARS-CoV-2, SARS-CoV, or MERS-CoV.

Embodiment 5. The method of embodiment 3, wherein the influenza virus is an influenza A virus.

Embodiment 6. The method of embodiment 1, wherein the subject is a human and has the infection or disease at the time of said administering.

Embodiment 7. The method of embodiment 1, wherein the subject is a human and has previously had the infection or disease at the time of said administering.

Embodiment 8. The method of embodiment 1, wherein the first particle and the second particle are administered intranasally.

Embodiment 9. The method of embodiment 1, wherein the first particle and the second particle are in powder form.

Embodiment 10. The method of embodiment 1, wherein the therapeutic agent is an antiviral, antibiotic, antifungal, antiparasitic, mucolytic, antibody, or vaccine.

Embodiment 11. The method of embodiment 10, wherein the antiviral is tamibarotene, remdesivir, clofazimine, ribavirin, oseltamivir, peramivir, zanamivir, baloxavir marboxil, chloroquine, favipiravir, triazavirin, umifenovir, or molnupiravir.

Embodiment 12. The method of embodiment 10, wherein the antibody is a neutralizing antibody, monoclonal antibody, antigen binding fragment (Fab) , or single chain variable fragment (scFv) .

Embodiment 13. The method of embodiment 10, wherein the vaccine is an mRNA vaccine, viral vector vaccine, inactivated vaccine, live-attenuated vaccine, or protein-based vaccine.

Embodiment 14. The method of embodiment 1, wherein the first particle deposits in the nasal cavity of the subject and the second particle deposits in the lung of the subject.

Embodiment 15. The method of embodiment 1, wherein the first particle has an aerodynamic diameter of about 10 μm to about 150 μm.

Embodiment 16. A composition of matter, comprising particles of two distinct sizes, wherein a first particle has an aerodynamic diameter of at least about 10 μm and a second particle has an aerodynamic diameter of about 1 μm to about 5 μm and the first and second particles comprise a therapeutic agent.

Embodiment 17. The composition of embodiment 16, wherein the first particle and the second particle are in powder form.

Embodiment 18. The composition of embodiment 16, wherein the therapeutic agent is an antiviral, antibiotic, antifungal, antiparasitic, mucolytic, antibody, or vaccine.

Embodiment 19. The composition of embodiment 18, wherein the antiviral is tamibarotene, remdesivir, clofazimine, ribavirin, oseltamivir, peramivir, zanamivir, baloxavir marboxil, chloroquine, favipiravir, triazavirin, umifenovir, or molnupiravir.

Embodiment 20. The composition of embodiment 16, wherein the first particle has an aerodynamic diameter of about 10 μm to about 150 μm.

Embodiment 21. The composition of embodiment 18, wherein the antibody is a neutralizing antibody, monoclonal antibody, Fab, or scFv.

Embodiment 22. The composition of embodiment 18, wherein the vaccine is an mRNA vaccine, viral vector vaccine, inactivated vaccine, live-attenuated vaccine, or protein-based vaccine

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Claims

A method for prophylactic or responsive treatment of a respiratory infection in a subject or disease of the respiratory tract of a subject or a symptom thereof, said method comprising administering a composition comprising an effective amount of a first particle with a median aerodynamic diameter of at least about 10 μm and a second particle with a median aerodynamic diameter of about 1 μm to about 5 μm, wherein the first particle and the second particle comprise a therapeutic agent.
The method of claim 1, wherein the respiratory infection is a viral infection or bacterial infection.
The method of claim 2, wherein the viral infection is a coronavirus infection, respiratory syncytial virus infection, or an influenza infection.
The method of claim 3, wherein the coronavirus is SARS-CoV-2, SARS-CoV, or MERS-CoV.
The method of claim 3, wherein the influenza virus is an influenza A virus.
The method of claim 2, wherein the bacterial infection is a Streptococcus pneumoniae infection or a Mycobacterium tuberculosis infection.
The method of claim 1, wherein the subject is a human and has the infection or disease at the time of said administering.
The method of claim 1, wherein the subject is a human and has previously had the infection or disease at the time of said administering.
The method of claim 1, wherein the first particle and the second particle are administered intranasally.
The method of claim 1, wherein the first particle and the second particle are in powder form.
The method of claim 1, wherein the therapeutic agent is an antiviral, antibiotic, antifungal, antiparasitic, mucolytic, antibody, or vaccine.
The method of claim 11, wherein the antiviral is tamibarotene, remdesivir, clofazimine, ribavirin, oseltamivir, peramivir, zanamivir, baloxavir marboxil, chloroquine, favipiravir, triazavirin, umifenovir, or molnupiravir.
The method of claim 11, wherein the antibody is a neutralizing antibody, monoclonal antibody, antigen binding fragment (Fab) , or single chain variable fragment (scFv) .
The method of claim 11, wherein the vaccine is an mRNA vaccine, viral vector vaccine, inactivated vaccine, live-attenuated vaccine, or protein-based vaccine.
The method of claim 1, wherein the first particle deposits in the nasal cavity of the subject and the second particle deposits in the lung of the subject.
The method of claim 1, wherein the first particle has an aerodynamic diameter of about 10 μm to about 150 μm.
A composition of matter, comprising particles of two distinct sizes, wherein a first particle has a median aerodynamic diameter of at least about 10 μm and a second particle has a median aerodynamic diameter of about 1 μm to about 5 μm and the first and second particles comprise a therapeutic agent.
The composition of claim 17, wherein the first particle and the second particle are in powder form.
The composition of claim 17, wherein the therapeutic agent is an antiviral, antibiotic, antifungal, antiparasitic, mucolytic, antibody, or vaccine.
The composition of claim 19, wherein the antiviral is tamibarotene, remdesivir, clofazimine, ribavirin, oseltamivir, peramivir, zanamivir, baloxavir marboxil, chloroquine, favipiravir, triazavirin, umifenovir, or molnupiravir.
The composition of claim 17, wherein the first particle has a median aerodynamic diameter of about 10 μm to about 150 μm.
The composition of claim 19, wherein the antibody is a neutralizing antibody, monoclonal antibody, Fab, or scFv.
The composition of claim 19, wherein the vaccine is an mRNA vaccine, viral vector vaccine, inactivated vaccine, live-attenuated vaccine, or protein-based vaccine.