Effect of lipid composition on RNA-Lipid nanoparticle properties and their sensitivity to thin-film freezing and drying.

A library of 16 lipid nanoparticle (LNP) formulations with orthogonally varying lipid molar ratios was designed and synthesized, using polyadenylic acid [poly(A)] as a model for mRNA, to explore the effect of lipid composition in LNPs on (i) the initial size of the resultant LNPs and encapsulation efficiency of RNA and (ii) the sensitivity of the LNPs to various conditions including cold storage, freezing (slow vs. rapid) and thawing, and drying. Least Absolute Shrinkage and Selection Operator (LASSO) regression was employed to identify the optimal lipid molar ratios and interactions that favorably affect the physical properties of the LNPs and enhance their stability in various stress conditions. LNPs exhibited distinct responses under each stress condition, highlighting the effect of lipid molar ratios and lipid interactions on the LNP physical properties and stability. It was then demonstrated that it is feasible to use thin-film freeze-drying to convert poly(A)-LNPs from liquid dispersions to dry powders while maintaining the integrity of the LNPs. Importantly, the residual moisture content in LNP dry powders significantly affected the LNP integrity.Residual moisture content of ≤ 0.5% or > 3-3.5% w/w negatively affected the LNP size and/or RNA encapsulation efficiency, depending on the LNP composition. Finally, it was shown that the thin-film freeze-dried LNP powders have desirable aerosol properties for potential pulmonary delivery. It was concluded that Design of Experiments can be applied to identify mRNA-LNP formulations with the desired physical properties and stability profiles. Additionally, optimizing the residual moisture content in mRNA-LNP dry powders during (thin-film) freeze-drying is crucial to maintain the physical properties of the LNPs.

Inhalable dry powders of microRNA-laden extracellular vesicles prepared by thin-film freeze-drying.

Extracellular vesicles (EVs) are endogenous vesicles that comprise a variety of submicron vesicular structures. Among these, exosomes have been widely investigated as delivery systems for small and large molecules. Herein, the thin-film freeze-drying technology was utilized to engineer aerosolizable dry powders of miR-335-laden induced EVs (iEV-335) generated in B cells for potential delivery into the lung to treat primary lung cancer and/or pulmonary metastases. The size distribution, structure, and morphology of iEV-335 were preserved after they were subjected to thin-film freeze-drying with the proper excipients. Importantly, iEV-335, in liquid or reconstituted from thin-film freeze-dried powders, were equally effective in downregulating SOX4 gene expression in LM2 human triple-negative mammary cancer cells. The iEV-335 dry powder compositions showed mass median aerodynamic diameters (MMAD) of around 1.2 µm with > 60 % of the emitted doses had an MMAD of ≤ 3 µm, indicating that the powders can potentially achieve efficient deposition within the alveolar region following oral inhalation, which is desirable for treatment of primary lung cancer and pulmonary metastases. Overall, it is concluded that it is feasible to apply thin-film freeze-drying to prepare aerosolizable dry powders of iEVs for pulmonary delivery.

The applications of Machine learning (ML) in designing dry powder for inhalation by using thin-film-freezing technology.

Dry powder inhalers (DPIs) are one of the most widely used devices for treating respiratory diseases. Thin--film--freezing (TFF) is a particle engineering technology that has been demonstrated to prepare dry powder for inhalation with enhanced physicochemical properties. Aerosol performance, which is indicated by fine particle fraction (FPF) and mass median aerodynamic diameter (MMAD), is an important consideration during the product development process. However, the conventional approach for formulation development requires many trial-and-error experiments, which is both laborious and time consuming. As a state-of-the art technique, machine learning has gained more attention in pharmaceutical science and has been widely applied in different settings. In this study, we have successfully built a prediction model for aerosol performance by using both tabular data and scanning electron microscopy (SEM) images. TFF technology was used to prepare 134 dry powder formulations which were collected as a tabular dataset. After testing many machine learning models, we determined that the Random Forest (RF) model was best for FPF prediction with a mean absolute error of ± 7.251%, and artificial neural networks (ANNs) performed the best in estimating MMAD with a mean absolute error of ± 0.393 µm. In addition, a convolutional neural network was employed for SEM image classification and has demonstrated high accuracy (>83.86%) and adaptability in predicting 316 SEM images of three different drug formulations. In conclusion, the machine learning models using both tabular data and image classification were successfully established to evaluate the aerosol performance of dry powder for inhalation. These machine learning models facilitate the product development process of dry powder for inhalation manufactured by TFF technology and have the potential to significantly reduce the product development workload. The machine learning methodology can also be applied to other formulation design and development processes in the future.

Formulation of dry powders of vaccines containing MF59 or AddaVax by Thin-Film Freeze-Drying: Towards a dry powder universal flu vaccine.

MF59® is an oil-in-water (O/W) nanoemulsion-based vaccine adjuvant that is often used in seasonal and pandemic influenza vaccines. We explored the feasibility of developing dry powders of vaccines adjuvanted with MF59 or AddaVax™, a preclinical grade equivalent of MF59 with the same composition and droplet size as MF59, by thin-film freeze-drying (TFFD). Liquid AddaVax alone was successfully converted to a dry powder by TFFD using trehalose as a stabilizing agent while maintaining the droplet size distribution of AddaVax after it was reconstituted. TFFD was then applied to convert liquid AddaVax-adjuvanted vaccines containing either a model antigen (e.g., ovalbumin) or mono-, bi-, and tri-valent recombinant hemagglutinin (rHA) protein-based H1 and/or H3 (universal) influenza vaccine candidates, as well as the MF59-containing Fluad® Quadrivalent influenza vaccine to dry powders. Both antigens and stabilizing agents affected the physical properties of the vaccines (e.g., mean particle size and particle size distribution) after the vaccines were subjected to TFFD. Importantly, the integrity and hemagglutination activity of the rHA antigens did not significantly change and the immunogenicity of reconstituted influenza vaccine candidates was maintained when evaluated in a mouse model. The vaccine dry powder was not sensitive to repeated freezing-and-thawing, in contrast to its liquid counterpart. It is concluded that TFFD can be applied to convert liquid vaccines containing MF59 or AddaVax to dry powders while maintaining the immunogenicity of the vaccines. Ultimately, TFFD technology may be used to prepare dry powders of multivalent universal influenza vaccines.

Dry powders for inhalation containing monoclonal antibodies made by thin-film freeze-drying.

Thin-film freeze-drying (TFFD) is a rapid freezing and then drying technique used to prepare inhalable dry powders from the liquid form for applications such as drug delivery to the lungs. Herein we report the preparation of aerosolizable dry powders of monoclonal antibodies (mAbs) by TFFD. We first formulated an IgG antibody with lactose/leucine (60:40, w/w) or trehalose/leucine (75:25) and tested their aerosol performance. The IgG 1% (w/w) formulated with lactose/leucine (60:40, w/w) in phosphate buffered saline (PBS) (IgG-1-LL-PBS) and processed by TFFD was found to produce the powder with desirable aerosol properties. We then replaced the IgG with anti-programmed cell death protein (anti-PD-1 mAb), a specific antibody, to prepare a dry powder (anti-PD1-1-LL-PBS), which performed similarly to the IgG-1-LL-PBS powder. The aerosol properties of the anti-PD1-1-LL-PBS dry powder were significantly better when TFFD was used to prepare the powder than when conventional shelf freeze-drying (shelf FD) was used. The TFFD dry powder had a porous structure with nanoaggregates and had a Tg value between 39 and 50 °C. When stored at room temperature, the anti-PD-1 mAb in the TFFD powder was more stable than that of the same formulation stored as a liquid. The addition of polyvinylpyrrolidone K40 in the formulation raised the Tg to 152 °C, which is expected to further increase the storage stability of the mAbs. The PD-1 binding activity of the anti-PD-1 mAbs after TFFD was not different from before TFFD. While protein loss, likely due to protein binding to vials and the thin-film freezing apparatus, was identified, we were able to minimize the loss by increasing the mAb concentration (i.e., from 1% to 13.2%). Micro-flow imaging revealed that the excipients and PBS affected subvisible aggregate formation. More subvisible mAb aggregates were generated when PBS was used, but the mAb content in the dry powders did not significantly affect the total subvisible aggregate count. Powders prepared with mannitol as an excipient showed the least amount of subvisible mAb aggregates. Finally, we showed that anti-TNF-α, another mAb, can also be converted to a dry powder with a similar composition by TFFD. We conclude that TFFD can be applied to produce stable, aerosolizable dry powders of mAbs for pulmonary delivery and that formulations must be optimized to maximize aerosol performance and minimize protein aggregation.

Niclosamide inhalation powder made by thin-film freezing: Multi-dose tolerability and exposure in rats and pharmacokinetics in hamsters.

In this work, we have developed and tested a dry powder form of niclosamide made by thin-film freezing (TFF) and administered it by inhalation to rats and hamsters to gather data about its toxicology and pharmacokinetics. Niclosamide, a poorly water-soluble drug, is an interesting drug candidate because it was approved over 60 years ago for use as an anthelmintic medication, but recent studies demonstrated its potential as a broad-spectrum antiviral with pharmacological effect against SARS-CoV-2 infection. TFF was used to develop a niclosamide inhalation powder composition that exhibited acceptable aerosol performance with a fine particle fraction (FPF) of 86.0% and a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 1.11 µm and 2.84, respectively. This formulation not only proved to be safe after an acute three-day, multi-dose tolerability and exposure study in rats as evidenced by histopathology analysis, and also was able to achieve lung concentrations above the required IC90 levels for at least 24 h after a single administration in a Syrian hamster model. To conclude, we successfully developed a niclosamide dry powder inhalation that overcomes niclosamide's limitation of poor oral bioavailability by targeting the drug directly to the primary site of infection, the lungs.

Aerosolizable siRNA-encapsulated solid lipid nanoparticles prepared by thin-film freeze-drying for potential pulmonary delivery.

Lipid nanoparticles are increasingly used for drug and gene delivery, including the delivery of small interfering RNA (siRNA). Pulmonary delivery of drug molecules carried by lipid nanoparticles directly into the lung may improve the treatment of certain lung diseases. The present study was designed to test the feasibility of engineering aerosolizable dry powder of lipid nanoparticles by thin-film freeze-drying (TFFD). Solid lipid nanoparticles (SLNs) comprised of lecithin, cholesterol, and a lipid-polyethylene glycol conjugate were prepared by solvent evaporation. Dry powders of the SLNs were prepared by TFFD, spray drying, or conventional shelf freeze-drying. The physical and aerosol properties of the dry powders as well as the physical properties of the SLNs reconstituted from the dry powders were evaluated. The particle size, polydispersity index, and the zeta potential of the SLNs were preserved after they were subjected to TFFD and reconstitution, but not after they were subjected to conventional shelf freeze-drying and reconstitution, and the dry powder prepared by TFFD showed better aerosol performance properties than that prepared by spray drying. SLNs encapsulated with siRNA can also be successfully transformed into aerosolizable dry powder by TFFD, and subjecting the siRNA-encapsulated SLNs to TFFD did not negatively affect the function of the siRNA. It is concluded that TFFD represents a promising method to prepare aerosolizable dry powder of lipid nanoparticles.

Development of Remdesivir as a Dry Powder for Inhalation by Thin Film Freezing.

Remdesivir exhibits in vitro activity against SARS-CoV-2 and was granted approval for emergency use. To maximize delivery to the lungs, we formulated remdesivir as a dry powder for inhalation using thin film freezing (TFF). TFF produces brittle matrix nanostructured aggregates that are sheared into respirable low-density microparticles upon aerosolization from a passive dry powder inhaler. In vitro aerodynamic testing demonstrated that drug loading and excipient type affected the aerosol performance of remdesivir. Remdesivir combined with optimal excipients exhibited desirable aerosol performance (up to 93.0% FPF< 5 µm; 0.82 µm mass median aerodynamic diameter). Remdesivir was amorphous after the TFF process, which benefitted drug dissolution in simulated lung fluid. TFF remdesivir formulations are stable after one month of storage at 25 °C/60% relative humidity. An in vivo pharmacokinetic evaluation showed that TFF remdesivir-leucine was poorly absorbed into systemic circulation while TFF remdesivir-Captisol® demonstrated increased systemic uptake compared to leucine. Remdesivir was hydrolyzed to the nucleoside analog GS-441524 in the lung, and levels of GS-441524 were greater in the lung with leucine formulation compared to Captisol®. In conclusion, TFF technology produces high-potency remdesivir dry powder formulations for inhalation that are suitable to treat patients with COVID-19 on an outpatient basis and earlier in the disease course where effective antiviral therapy can reduce related morbidity and mortality.

Formulation strategies in immunotherapeutic pharmaceutical products.

Development of immunologic-based biopharmaceutical products have strikingly increased in recent years and have made evident contributions to human health. Antibodies are the leading entity in immunotherapy, while chimeric antigen receptor T cells therapies are the advent of a novel strategy in this area. In order to enable antibody candidates or cells available as products, formulation is critical in terms of stabilize molecules or cells to achieve practical shelf life, storage and handling conditions. Here we provide a concise and contemporary review of ongoing formulation strategies and excipients used in approved antibodies and cellular therapeutic products. Excipients are categorized, and their function in formulations are discussed.

Influence of Carbamazepine Dihydrate on the Preparation of Amorphous Solid Dispersions by Hot Melt Extrusion.

Amorphous solid dispersions (ASDs) are commonly used in the pharmaceutical industry to improve the dissolution and bioavailability of poorly water-soluble drugs. Hot melt extrusion (HME) has been employed to prepare ASD based products. However, due to the narrow processing window of HME, ASDs are normally obtained with high processing temperatures and mechanical stress. Interestingly, one-third of pharmaceutical compounds reportedly exist in hydrate forms. In this study, we selected carbamazepine (CBZ) dihydrate to investigate its solid-state changes during the dehydration process and the impact of the dehydration on the preparation of CBZ ASDs using a Leistritz micro-18 extruder. Various characterization techniques were used to study the dehydration kinetics of CBZ dihydrate under different conditions. We designed the extrusion runs and demonstrated that: 1) the dehydration of CBZ dihydrate resulted in a disordered state of the drug molecule; 2) the resulted higher energy state CBZ facilitated the drug solubilization and mixing with the polymer matrix during the HME process, which significantly decreased the required extrusion temperature from 140 to 60 °C for CBZ ASDs manufacturing compared to directly processing anhydrous crystalline CBZ. This work illustrated that the proper utilization of drug hydrates can significantly improve the processability of HME for preparing ASDs.

A Repurposed Drug for Brain Cancer: Enhanced Atovaquone Amorphous Solid Dispersion by Combining a Spontaneously Emulsifying Component with a Polymer Carrier.

Glioblastoma multiforme (GBM) is the most common and lethal central nervous system tumor. Recently, atovaquone has shown inhibition of signal transducer and activator transcription 3, a promising target for GBM therapy. However, it is currently unable to achieve therapeutic drug concentrations in the brain with the currently reported and marketed formulations. The present study sought to explore the efficacy of atovaquone against GBM as well as develop a formulation of atovaquone that would improve oral bioavailability, resulting in higher amounts of drug delivered to the brain. Atovaquone was formulated as an amorphous solid dispersion using an optimized formulation containing a polymer and a spontaneously emulsifying component (SEC) with greatly improved wetting, disintegration, dispersibility, and dissolution properties. Atovaquone demonstrated cytotoxicity against GBM cell lines as well as provided a confirmed target for atovaquone brain concentrations in in vitro cell viability studies. This new formulation approach was then assessed in a proof-of-concept in vivo exposure study. Based on these results, the enhanced amorphous solid dispersion is promising for providing therapeutically effective brain levels of atovaquone for the treatment of GBM.

Flocculated amorphous nanoparticles for highly supersaturated solutions.

PURPOSE: To recover polymer-stabilized amorphous nanoparticles from aqueous dispersions efficiently by salt flocculation and to show that the particles redisperse and dissolve rapidly to produce highly supersaturated solutions. METHODS: Nanoparticle dispersions of itraconazole stabilized by nonionic polymers were formed by antisolvent precipitation and immediately flocculated with sodium sulfate, filtered and dried. The size after redispersion in water, crystallinity, and morphology were compared with those for particles produced by spray drying and rapid freezing. RESULTS: Particle drug loading increased to approximately 90% after salt flocculation and removal of excess polymer with the filtrate. The formation of the flocs at constant particle volume fraction led to low fractal dimensions (open flocs), which facilitated redispersion in water to the original primary particle size of approximately 300 nm. Amorphous particles, which were preserved throughout the flocculation-filtration-drying process, dissolved to supersaturation levels of up to 14 in pH 6.8 media. In contrast, both spray dried and rapidly frozen nanoparticle dispersions crystallized and did not produce submicron particle dispersions upon addition to water, nor high supersaturation values. CONCLUSIONS: Salt flocculation produces large yields of high surface area amorphous nanoparticle powders that de-aggregate and dissolve rapidly upon redispersion in pH 6.8 media, for supersaturation levels up to 14.