Intranasal delivery of thin-film freeze-dried monoclonal antibodies using a powder nasal spray system.

Monoclonal antibodies (mAbs) administered intranasally as dry powders can be potentially applied for the treatment or pre-exposure prevention of viral infections in the upper respiratory tract. However, a method to transform the mAbs from liquid to dry powders suitable for intranasal administration and a device that can spray the dry powders to the desired region of the nasal cavity are needed to fully realize the potentials of the mAbs. Herein, we report that thin-film freeze-dried mAb powders can be sprayed into the posterior nasal cavity using Aptar Pharma's Unidose (UDS) Powder Nasal Spray System. AUG-3387, a human-derived mAb that neutralizes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was used in the present study. First, we prepared thin-film freeze-dried AUG-3387 powders (i.e., TFF AUG-3387 powders) from liquid formulations containing different levels of mAbs. The TFF AUG-3387 powder with the highest solid content (i.e., TFF AUG-3387C) was then chosen for further characterization, including the evaluation of the plume geometry, spray pattern, and particle size distribution after the powder was sprayed using the UDS Powder Nasal Spray. Finally, the deposition patterns of the TFF AUG-3387C powder sprayed using the UDS Powder delivery system were studied using 3D-printed nasal replica casts based on the CT scans of an adult and a child. It is concluded that it is feasible to intranasally deliver mAbs as dry powders by transforming the mAbs into dry powders using thin-film freeze-drying and then spraying the powder using a powder nasal spray system.

Characterization of mRNA Lipid Nanoparticles by Electron Density Mapping Reconstruction: X-ray Scattering with Density from Solution Scattering (DENSS) Algorithm.

PURPOSE: This study aimed to test the feasibility of using Small Angle X-ray Scattering (SAXS) coupled with Density from Solution Scattering (DENSS) algorithm to characterize the internal architecture of messenger RNA-containing lipid nanoparticles (mRNA-LNPs). METHODS: The DENSS algorithm was employed to construct a three-dimensional model of average individual mRNA-LNP. The reconstructed models were cross validated with cryogenic transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS) to assess size, morphology, and internal structure. RESULTS: Cryo-TEM and DLS complemented SAXS, revealed a core-shell mRNA-LNP structure with electron-rich mRNA-rich region at the core, surrounded by lipids. The reconstructed model, utilizing the DENSS algorithm, effectively distinguishes mRNA and lipids via electron density mapping. Notably, DENSS accurately models the morphology of the mRNA-LNPs as an ellipsoidal shape with a "bleb" architecture or a two-compartment structure with contrasting electron densities, corresponding to mRNA-filled and empty lipid compartments, respectively. Finally, subtle changes in the LNP structure after three freeze-thaw cycles were detected by SAXS, demonstrating an increase in radius of gyration (Rg) associated with mRNA leakage. CONCLUSION: Analyzing SAXS profiles based on DENSS algorithm to yield a reconstructed electron density based three-dimensional model can be a useful physicochemical characterization method in the toolbox to study mRNA-LNPs and facilitate their development.

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.

Feasibility of intranasal delivery of thin-film freeze-dried, mucoadhesive vaccine powders.

Intranasal vaccination by directly applying a vaccine dry powder is appealing. However, a method that can be used to transform a vaccine from a liquid to a dry powder and a device that can be used to administer the powder to the desired region(s) of the nasal cavity are critical for successful intranasal vaccination. In the present study, using a model vaccine that contains liposomal monophosphoryl lipid A and QS-21 adjuvant (AdjLMQ) and ovalbumin (OVA) as a model antigen, it was shown that thin-film freeze-drying can be applied to convert the liquid vaccine containing sucrose at a sucrose to lipid ratio of 15:1 (w/w) into dry powders, in the presence or absence of carboxymethyl cellulose sodium salt (CMC) as a mucoadhesive agent. Ultimately, the thin-film freeze-dried AdjLMQ/OVA vaccine powder containing 1.9% (w/w) of CMC (i.e., TFF AdjLMQ/OVA/CMC1.9% powder) was selected for additional evaluation because the TFF AdjLMQ/OVA/CMC1.9% powder was mucoadhesive and maintained the integrity of the antigen and the physical properties of the vaccine. Compared to the TFF AdjLMQ/OVA powder that did not contain CMC, the TFF AdjLMQ/OVA/CMC1.9% powder had a lower moisture content and a higher glass transition temperature. In addition, the TFF AdjLMQ/OVA/CMC1.9% thin films were relatively thicker than the TFF AdjLMQ/OVA thin films without CMC. When sprayed with Aptar Pharma's Unidose Powder Nasal Spray System (UDSP), the TFF AdjLMQ/OVA powder and the TFF AdjLMQ/OVA/CMC1.9% powder generated similar particle size distribution curves, spray patterns, and plume geometries. Importantly, after the TFF AdjLMQ/OVA/CMC1.9% powder was sprayed with the UDSP nasal device, the integrity of the OVA antigen and the AdjLMQ liposomes did not change. Finally, a Taguchi L4 orthogonal array was applied to identify the optimal parameters for using the UDSP device to deliver the TFF AdjLMQ/OVA/CMC1.9% powder to the middle and lower turbinate and the nasopharynx regions in both adult and child nasal replica casts. Results from this study showed that it is feasible to apply the thin-film freeze-drying technology to transform a nasal vaccine candidate from liquid to a dry powder and then use the UDSP nasal device to deliver the vaccine powder to the desired regions in the nasal cavity for intranasal vaccination.

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.

Development of (Inhalable) Dry Powder Formulations of AS01B-Containing Vaccines Using Thin-Film Freeze-Drying.

AS01B is a liposomal formulation of two immunostimulants namely 3-O-desacyl-4́-monophosphoryl lipid A (MPL) and QS-21. The liposomal formulation of AS01B reduces the endotoxicity of MPL and the lytic activity of QS-21. The AS01B-adjuvanted Shingrix vaccine is marketed in a two-vial presentation, with the liquid AS01B liposomes in one vial and the antigen as a dry powder in another vial. In the present study, we tested the feasibility of applying thin-film freeze-drying (TFFD) to engineer dry powders of the AS01B liposomal adjuvant alone or vaccines containing AS01B as an adjuvant. Initially, we showed that after the AS01B liposomal adjuvant was subjected to TFFD using sucrose as a stabilizer at 4% w/v, the particle size distribution of AS01B liposomes reconstituted from the dry powder was identical to the liquid adjuvant before drying. We then showed using ovalbumin (OVA) as a model antigen adjuvanted with AS01B (AS01B/OVA) that subjecting the AS01B/OVA vaccine to TFFD and subsequent reconstitution did not negatively affect the AS01B liposome particle size, nor the immunogenicity of the vaccine. Importantly, the thin-film freeze-dried AS01B/OVA vaccine, unlike its liquid counterpart, was not sensitive to repeated freezing-and-thawing. The developed AS01B/OVA dry powder also showed the desirable aerosol properties (i.e., fine particle fraction of 66.3 ± 4.9% and mass median aerodynamic diameter of 2.4 ± 0.1 µm) for potential pulmonary administration. Finally, the feasibility of using TFFD to prepare dry powders of AS01B-adjuvanted vaccines was further confirmed using AS01B-adjuvanted Fluzone Quadrivalent and Shingrix, which contains AS01B. It is concluded that the TFFD technology can enable the formulation of AS01B-adjuvanted vaccines as freezing-insensitive, inhalable dry powders in a single-vial presentation.

Next-Generation COVID-19 Vaccines Should Take Efficiency of Distribution into Consideration.

The dire need for safe and effective coronavirus disease (COVID-19) vaccines is met with many vaccine candidates being evaluated in pre-clinical and clinical studies. The COVID-19 vaccine candidates currently in phase 3 or phase 2/3 clinical trials as well as those that recently received emergency use authorization (EUA) from the United States Food and Drug Administration (FDA) and/or other regulatory agencies worldwide require either cold (i.e., 2-8°C) or even freezing temperatures as low as -70°C for storage and distribution. Thus, existing cold chain will struggle to support both the standard national immunization programs and COVID-19 vaccination. The requirement for cold chain is now a major challenge towards worldwide rapid mass vaccination against COVID-19. In this commentary, we stress that thermostabilizing technologies are available to enable cold chain-free vaccine storage and distribution, as well as potential needle-free vaccination. Significant efforts on thermostabilizing technologies must now be applied on next-generation COVID-19 vaccines for more cost-effective worldwide mass vaccination and COVID-19 eradication.

Novel formulations and drug delivery systems to administer biological solids.

Recent advances in formulation sciences have expanded the previously limited design space for biological modalities, including peptide, protein, and vaccine products. At the same time, the discovery and application of new modalities, such as cellular therapies and gene therapies, have presented formidable challenges to formulation scientists. We explore these challenges and highlight the opportunities to overcome them through the development of novel formulations and drug delivery systems as biological solids. We review the current progress in both industry and academic laboratories, and we provide expert perspectives in those settings. Formulation scientists have made a tremendous effort to accommodate the needs of these novel delivery routes. These include stability-preserving formulations and dehydration processes as well as dosing regimes and dosage forms that improve patient compliance.

The safety, efficacy and pharmaceutical quality of male enhancement nutraceuticals bought online: Truth versus claim.

OBJECTIVE: Nutraceutical products are widely used for their claimed therapeutic benefits. However, falsified or adulterated nutraceuticals present a major health threat to consumers. This study investigates the pharmaceutical quality, safety and anti-inflammatory effects of six male enhancement nutraceuticals that claim to be 100% natural. METHODS: Three batches of six male enhancement products were tested to detect the presence and levels of adulterants via high-performance liquid chromatography (HPLC). The pharmaceutical quality of the selected nutraceuticals was tested with near infrared spectroscopy (NIR) and SeDeM. The cytotoxic effects of these products on HepG2 cells were determined through cell proliferation (XTT) and lactate dehydrogenase (LDH) cytotoxicity assays. Lastly, the in vitro inflammatory effects of these products were investigated using murine J774 macrophages through cytokine release analysis. RESULTS: HPLC analysis detected the presence of sildenafil citrate, a vasodilator, and the active ingredient in Viagra and Revatio, in all batches of the products we analyzed. Amount of sildenafil citrate ranged from 0.45 mg to 51.85 mg among different batches. NIR assessment showed inter- and intra-batch heterogeneity in product composition. Results of the XTT and LDH assays showed significant cytotoxic effects of the analyzed products. XTT analysis revealed that the viability of HepG2 treated with tested products varied from 27.57% to 41.43%. Interestingly, the male enhancement products also showed anti-inflammatory effects. CONCLUSION: Despite their labeling as 100% natural, all products tested in this study contained levels of sildenafil citrate, which was not reported on the packaging. There was a lack of pharmaceutical uniformity among products of the same batch and across different batches. Additionally, the products we tested had cytotoxic effects. These study findings highlight the adulteration, poor quality and hazard of these nutraceuticals. Therefore, strict regulation of these products and standardization of the definition of nutraceuticals are urgently needed. Further, these falsely advertised products should be withdrawn from the market due to potential adverse effects on the health of their consumers.

Amorphous solid dispersion dry powder for pulmonary drug delivery: Advantages and challenges.

Amorphous solid dispersion (ASD) is commonly used in pharmaceutical industry. It has been mainly employed to enhance the oral bioavailability of poorly water-soluble drugs that belong to class II and IV of the biopharmaceutical classification system but has showed promise in other areas of pharmaceutical research. In this review, the potential and limitations of ASD dry powder for inhalation are discussed. ASD powder for inhalation (ASD-IP) is commonly prepared by spray drying technique. The physicochemical characteristics of ASD-IP could be tailored to achieve effective lung deposition. ASD-IP could also attain rapid dissolution behavior to achieve therapeutically effective concentration either locally or systemically before particle clearance in the lung. The key challenges of using ASD powder for inhalation include the possible chemical and/or physical instability of the amorphous phase during manufacturing and in vivo, and the moisture and temperature sensitivity of ASD-IP that affects its storage stability.

Silent bleeding in children and adolescents with immune thrombocytopenia: relation to laboratory parameters and health related quality of life.

Occult hemorrhage can occur in any internal organ in ITP patients. Four sites of occult hemorrhage require attention including microscopic hematuria, fecal occult blood loss, retinal hemorrhage, and silent intracranial hemorrhage. The aim of this study was to investigate the frequency of subclinical bleeding in children with ITP and its relation to clinical and laboratory disease parameters including bleeding score and health related quality of life. This cross-sectional study included 40 ITP patients recruited from the Pediatric Hematology/Oncology unit, Children's Hospital, Ain Shams University, Cairo, Egypt. Inclusion criteria were patients with ITP (acute, persistent or chronic) having platelet count of 20,000/cmm or less at diagnosis/relapse, patients with overt bleeding and patients with secondary ITP were excluded. Occult blood in stools and urine analysis, fundus examination, and non-contrast brain MRI for microbleeds were done. Out of the forty included patients, 24 had chronic, 11 had acute and 5 had persistent ITP. Eleven patients had occult bleeds. Two patients had occult blood in stools, five had microscopic hematuria, one had retinal bleeds and three patients had brain microbleeds. Their mean age was 10.23 ± 4.18 years and their mean initial bleeding score was 2.55 ± 0.82. Nine patients with occult bleeding were chronic, one persistent and one acute ITP patients. There were no significant differences between patients with occult bleeding and those without as regards the initial bleeding score, platelet counts and hemoglobin level, as well as the mean platelet counts and mean hemoglobin level over the disease duration (p > 0.5). The scoring of the parent's life, Child and parents' quality of life was low in 3 out of 11 patients with occult bleeding. There was no significant difference between patients with occult bleeding and those without as regards the ITP child and parents' quality of life items (p = 0.850 and 0.511 respectively). Our results suggest that subclinical bleeding is a potential risk in children with ITP, more commonly chronic ITP patients. We could not demonstrate a significant relation of occult bleeding to the laboratory findings, bleeding score, and the ITP health quality of life; nevertheless, the significance of the routine assessment of occult bleeding in ITP and the identification of high-risk patients require additional studies.

A Self-Nanoemulsifying Drug Delivery System for Enhancing the Oral Bioavailability of Candesartan Cilexetil: Ex Vivo and In Vivo Evaluation.

The drug delivery of candesartan cilexetil encounters an obstacle of low absolute oral bioavailability which is attributed mainly to its low aqueous solubility and efflux by intestinal P-glycoprotein (P-gp) transporters. However, the extent of P-gp contribution in the reduced oral bioavailability of candesartan cilexetil is not clear. In this study, a previously developed candesartan cilexetil-loaded self-nanoemulsifying drug delivery system (SNEDDS) was evaluated for its ability to increase the drug oral bioavailability via the inhibition of intestinal P-gp transporters. Despite the developed SNEDDS showing P-gp inhibition activity, P-gp-mediated efflux was found to have a minor role in the reduced oral bioavailability of candesartan cilexetil. On the other hand, the high surfactant concentration used in SNEDDS formulation represents a major challenge toward their widespread application especially for chronically administered drugs. The designed acute and subacute toxicity studies revealed that the degree of intestinal mucosal damage decreases as the treatment period increases. The latter observation was attributed to the reversibility of surfactant-induced mucosal damage. Thus, the developed SNEDDS could be considered as a promising delivery system for enhancing the oral bioavailability of chronically administered drugs.

Self-emulsifying drug-delivery systems modulate P-glycoprotein activity: role of excipients and formulation aspects.

Self-emulsifying drug-delivery systems (SEDDS) have been widely employed to ameliorate the oral bioavailability of P-glycoprotein (P-gp) substrate drugs and to overcome multidrug resistance in cancer cells. However, the role of formulation aspects in the reduced P-gp activity is not fully understood. In this review, we first explore the role of various SEDDS excipients in the reduced P-gp activity with the main emphasis on the effective excipient concentration range for excipient-mediated modulation of P-gp activity and then we discuss the synergistic effect of various formulation aspects on the excipient-mediated modulation of P-gp activity. This review provides an approach to develop a rationally designed SEDDS to overcome P-gp-mediated drug efflux.

Role of self-emulsifying drug delivery systems in optimizing the oral delivery of hydrophilic macromolecules and reducing interindividual variability.

Self-emulsifying drug delivery systems (SEDDS) have been widely employed to improve the oral bioavailability of poorly soluble drugs. In the past few years, SEDDS were extensively investigated to overcome various barriers encountered in the oral delivery of hydrophilic macromolecules (e.g., protein/peptide therapeutics and plasmid DNA (pDNA)), as well as in lowering the effect of food on drugs' bioavailability. However, the main mechanism(s) by which SEDDS could achieve such promising effects remains not fully understood. This review summarizes the recent progress in the use of SEDDS for protecting protein therapeutics and/or pDNA against enzymatic degradation and increasing the oral bioavailability of various drug substances regardless of the dietary condition. Understanding the underlying mechanism(s) of such promising applications will aid in the future development of rationally designed SEDDS. Entrapment of hydrophilic macromolecules in the oil phase of the formed emulsion is critical for protection of the loaded cargoes against enzymatic degradation and the enhancement of oral bioavailability. On the other hand, drug administration as a preconcentrated solution in the SEDDS preconcentrate allows the process of drug absorption to occur independently of the dietary condition, and thus reducing interindividual variability that results from concomitant food intake.

Development and in vitro/in vivo performance of self-nanoemulsifying drug delivery systems loaded with candesartan cilexetil.

Candesartan cilexetil is widely used in the management of hypertension and heart failure. The drug delivery encounters obstacles of poor aqueous solubility, efflux by intestinal P-glycoprotein and vulnerability to enzymatic degradation in small intestine. Self-nanoemulsifying drug delivery systems (SNEDDS) loaded with candesartan cilexetil were successfully developed to overcome such obstacles. Preliminary screening was carried out to select proper surfactant, co-surfactant and oil combination for successful SNEDDS formulation. All screened excipients were reported for their P-glycoprotein and cytochrome P450 3A4 (CYP3A4) modulation activity. Ternary and pseudo ternary diagrams were constructed to optimize the system. Peppermint oil and clove oil showed a high emulsification ability. The nature of obtained dispersions was identified to be nanoemulsions. Twenty-four formulations were evaluated for stability, robustness to dilution and self-emulsification efficiency. All formulations showed a very short emulsification time of <2min. The emulsification efficiency was significantly superior at pH6.8, at which the largest self-emulsifying region was also observed. Eight formulations were selected for further characterization according to cloud point measurement; mean droplet size, poly dispersity index (PDI) and zeta potential determination in addition to in vitro drug release study. All selected formulations showed very high cloud points (70-90°C), ultrafine mean droplet size (12±1.4 to 24.5±2.13nm), very low PDI values (0.015-0.1305) and almost a complete drug release after 12h. Formulation F15 (Peppermint oil 55% w/w: Cremophor RH40 25% w/w: Labrasol 20% w/w) was selected for further characterization. Its droplet size showed robustness to different dilution folds with different media and its TEM photograph showed spherical particles without any apparent aggregation even after 24h. Formulation F15 successfully controlled the systolic blood pressure of hypertensive rats for 24h with the maximum effect was observed after 2h. These results indicate that, SNEDDS could be promising delivery systems with a rapid onset of action and prolonged therapeutic effect of candesartan cilexetil.