Objective monitoring tools for improved management of childhood asthma.

Asthma is a common chronic disease amongst children. Epidemiological studies showed that the mortality rate of asthma in children is still high worldwide. Asthma control is therefore essential to minimize asthma exacerbations, which can be fatal if the condition is poorly controlled. Frequent monitoring could help to detect asthma progression and ensure treatment effectiveness. Although subjective asthma monitoring tools are available, the results vary as they rely on patients' self-perception. Emerging evidence suggests several objective tools could have the potential for monitoring purposes. However, there is no consensus to standardise the use of objective monitoring tools. In this review, we start with the prevalence and severity of childhood asthma worldwide. Then, we detail the latest available objective monitoring tools, focusing on their effectiveness in paediatric asthma management. Publications of spirometry, fractional exhaled nitric oxide (FeNO), hyperresponsiveness tests and electronic monitoring devices (EMDs) between 2016 and 2023 were included. The potential advantages and limitations of each tool were also discussed. Overall, this review provides a summary for researchers dedicated to further improving objective paediatric asthma monitoring and provides insights for clinicians to incorporate different objective monitoring tools in clinical practices.

Co-Delivery of D-LAK Antimicrobial Peptide and Capreomycin as Inhaled Powder Formulation to Combat Drug-Resistant Tuberculosis.

INTRODUCTION: The emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) posed a severe challenge to tuberculosis (TB) management. The treatment of MDR-TB involves second-line anti-TB agents, most of which are injectable and highly toxic. Previous metabolomics study of the Mtb membrane revealed that two antimicrobial peptides, D-LAK120-A and D-LAK120-HP13, can potentiate the efficacy of capreomycin against mycobacteria. AIMS: As both capreomycin and peptides are not orally available, this study aimed to formulate combined formulations of capreomycin and D-LAK peptides as inhalable dry powder by spray drying. METHODS AND RESULTS: A total of 16 formulations were prepared with different levels of drug content and capreomycin to peptide ratios. A good production yield of over 60% (w/w) was achieved in most formulations. The co-spray dried particles exhibited spherical shape with a smooth surface and contained low residual moisture of below 2%. Both capreomycin and D-LAK peptides were enriched at the surface of the particles. The aerosol performance of the formulations was evaluated with Next Generation Impactor (NGI) coupled with Breezhaler®. While no significant difference was observed in terms of emitted fraction (EF) and fine particle fraction (FPF) among the different formulations, lowering the flow rate from 90 L/min to 60 L/min could reduce the impaction at the throat and improve the FPF to over 50%. CONCLUSIONS: Overall, this study showed the feasibility of producing co-spray dried formulation of capreomycin and antimicrobial peptides for pulmonary delivery. Future study on their antibacterial effect is warranted.

Spray-Dried and Spray-Freeze-Dried Powder Formulations of an Anti-Interleukin-4Rα Antibody for Pulmonary Delivery.

OBJECTIVE: The therapeutic options for severe asthma are limited, and the biological therapies are all parenterally administered. The purpose of this study was to formulate a monoclonal antibody that targets the receptor for IL-4, an interleukin implicated in the pathogenesis of severe asthma, into a dry powder intended for delivery via inhalation. METHODS: Dehydration was achieved using either spray drying or spray freeze drying, which exposes the thermolabile biomacromolecules to stresses such as shear and adverse temperatures. 2-hydroxypropyl-beta-cyclodextrin was incorporated into the formulation as protein stabiliser and aerosol performance enhancer. The powder formulations were characterised in terms of physical and aerodynamic properties, while the antibody was assessed with regard to its structural stability, antigen-binding ability, and in vitro biological activity after drying. RESULTS: The spray-freeze-dried formulations exhibited satisfactory aerosol performance, with emitted fraction exceeding 80% and fine particle fraction of around 50%. The aerosolisation of the spray-dried powders was hindered possibly by high residual moisture. Nevertheless, the antigen-binding ability and inhibitory potency were unaffected for the antibody in the selected spray-dried and spray-freeze-dried formulations, and the antibody was physically stable even after one-year storage at ambient conditions. CONCLUSIONS: The findings of this study establish the feasibility of developing an inhaled dry powder formulation of an anti-IL-4R antibody using spray drying and spray freeze drying techniques with potential for the treatment of severe asthma.

Optimization of PEGylated KL4 Peptide for siRNA Delivery with Improved Pulmonary Tolerance.

Pulmonary delivery of small interfering RNA (siRNA) is a promising therapeutic strategy for treating various respiratory diseases but an effective carrier for the delivery of siRNA into the cells of the lungs and a robust gene-silencing effect is still lacking. Previously, we reported that the KL4 peptide, a synthetic cationic peptide with a repeating KLLLL sequence, can mediate effective siRNA transfection in lung epithelial cells but its high hydrophobic leucine content, and hence poor water solubility, limits its application as a delivery vector. Here, we show that the covalent attachment of monodisperse poly(ethylene glycol) (PEG) improves the solubility of KL4 and the uptake of its complex with siRNA into lung epithelial cells, such that very robust silencing is produced. All PEGylated KL4 peptides, with PEG length varying between 6 and 24 monomers, could bind and form nanosized complexes with siRNA, but the interaction between siRNA and peptides became weaker as the PEG chain length increased. All PEGylated KL4 peptides exhibited satisfactory siRNA transfection efficiency on three human lung epithelial cell lines, including A549 cells, Calu-3 cells, and BEAS-2B cells. The PEG12KL4 peptide, which contains 12 monomers of PEG, was optimal for siRNA delivery and also demonstrated a low risk of inflammatory response and toxicity in vivo following pulmonary administration.

From Pulmonary Surfactant, Synthetic KL4 Peptide as Effective siRNA Delivery Vector for Pulmonary Delivery.

Pulmonary delivery of small interfering RNA (siRNA) has huge potential for the treatment of a wide range of respiratory diseases. The ability of naked siRNA to transfect cells in the lungs without a delivery vector has prompted the investigation of whether an endogenous component is at least partially responsible for the cellular uptake of siRNA, and whether a safe and efficient delivery system could be developed from this component to further improve the transfection efficiency. Surfactant protein B (SP-B), a positively charged protein molecule found in lung surfactant, is one of the possible candidates. While the role of SP-B in siRNA transfection remains to be determined, the SP-B mimic, synthetic KL4 peptide, was investigated in this study as a potential siRNA carrier. KL4 is a 21-residue cationic peptide that was able to bind to siRNA to form nanosized complexes. It mediated siRNA transfection effectively in vitro on human lung epithelial cells, A549 cells, and BEAS-2B cells, which was comparable to Lipofectamine 2000. When commercial pulmonary surfactant (Infasurf) was added in the transfection medium, the gene silencing effect of siRNA in cells transfected with Lipofectamine 2000 was completely abolished, whereas those transfected with KL4 remained unaffected. At 4 °C, KL4 failed to deliver siRNA into the cells, indicating that an energy-dependent process was involved in the uptake of the complexes. Chlorpromazine (inhibitor of chathrin-mediated endocytosis), but not nystatin (inhibitor of caveolae-mediated endocytosis), inhibited the uptake of KL4/siRNA complexes, suggesting that they entered cells through clathrin-mediated endocytosis. There was no sign of cytotoxicity or immune response caused by KL4 and KL4/siRNA complexes. Overall, this study demonstrated that synthetic KL4 peptide is a promising candidate for siRNA carrier for pulmonary delivery and could be a potential platform for delivering other types of nucleic acid therapeutics.

Incorporation of a Nuclear Localization Signal in pH Responsive LAH4-L1 Peptide Enhances Transfection and Nuclear Uptake of Plasmid DNA.

The major intracellular barriers associated with DNA delivery using nonviral vectors are inefficient endosomal/lysosomal escape and poor nuclear uptake. LAH4-L1, a pH responsive cationic amphipathic peptide, is an efficient DNA delivery vector that promotes the release of nucleic acid into cytoplasm through endosomal escape. Here we further enhance the DNA transfection efficiency of LAH4-L1 by incorporating nuclear localizing signal (NLS) to promote nuclear importation. Four NLSs were investigated: Simian virus 40 (SV40) large T-antigen derived NLS, nucleoplasmin targeting signal, M9 sequence, and the reverse SV40 derived NLS. All peptides tested were able to form positively charged nanosized complexes with DNA. Significant improvement in DNA transfection was observed in slow-dividing epithelial cancer cells (Calu-3), macrophages (RAW264.7), dendritic cells (JAWSII), and thymidine-induced growth-arrested cells, but not in rapidly dividing cells (A549). Among the four NLS-modified peptides, PK1 (modified with SV40 derived NLS) and PK2 (modified with reverse SV40 derived NLS) were the most consistent in improving DNA transfection; up to a 10-fold increase in gene expression was observed for PK1 and PK2 over the unmodified LAH4-L1. Additionally PK1 and PK2 were shown to enhance cellular uptake as well as nuclear entry of DNA. Overall, we show that the incorporation of SV40 derived NLS, in particular, to LAH4-L1 is a promising strategy to improve DNA delivery efficiency in slow-dividing cells and dendritic cells, with development potential for in vivo applications and as a DNA vaccine carrier.

Delivery of RNAi Therapeutics to the Airways-From Bench to Bedside.

RNA interference (RNAi) is a potent and specific post-transcriptional gene silencing process. Since its discovery, tremendous efforts have been made to translate RNAi technology into therapeutic applications for the treatment of different human diseases including respiratory diseases, by manipulating the expression of disease-associated gene(s). Similar to other nucleic acid-based therapeutics, the major hurdle of RNAi therapy is delivery. Pulmonary delivery is a promising approach of delivering RNAi therapeutics directly to the airways for treating local conditions and minimizing systemic side effects. It is a non-invasive route of administration that is generally well accepted by patients. However, pulmonary drug delivery is a challenge as the lungs pose a series of anatomical, physiological and immunological barriers to drug delivery. Understanding these barriers is essential for the development an effective RNA delivery system. In this review, the different barriers to pulmonary drug delivery are introduced. The potential of RNAi molecules as new class of therapeutics, and the latest preclinical and clinical studies of using RNAi therapeutics in different respiratory conditions are discussed in details. We hope this review can provide some useful insights for moving inhaled RNAi therapeutics from bench to bedside.

Oleanolic Acid Loaded PEGylated PLA and PLGA Nanoparticles with Enhanced Cytotoxic Activity against Cancer Cells.

Oleanolic acid (OA) is a natural triterpenoid with anticancer properties, but its hydrophobic nature and poor aqueous solubility pose challenges in pharmaceutical formulation development. The present study aimed at developing OA-loaded mPEG-PLGA or mPEG-PLA nanoparticles (NPs) to improve the delivery of OA. The NPs were prepared by nanoprecipitation, and their physicochemical properties were characterized. The OA encapsulation efficiency of the NPs was between 40 and 75%. The size of the OA-loaded NPs was around 200-250 nm, which fell within the range required for tumor targeting by means of the enhanced permeability and retention (EPR) effect, and the negatively charged NPs remained physically stable for over 20 weeks with no aggregation observed. The OA-loaded NPs produced significant cytotoxic effects through apoptosis in cancer cell lines. Overall, the OA-loaded mPEG-PLGA NPs and mPEG-PLA NPs shared similar physicochemical properties. The former, especially the OA-loaded mPEG-P(D,L)LGA NPs, were more cytotoxic to cancer cells and therefore were more efficient for OA delivery.

Inhalable dry powder formulations of siRNA and pH-responsive peptides with antiviral activity against H1N1 influenza virus.

Pulmonary delivery of siRNA has considerable therapeutic potential for treating viral respiratory infectious diseases including influenza. By introducing siRNA that targets the conserved region of viral genes encoding nucleocapsid protein (NP), viral mRNAs can be degraded and viral replication can be inhibited in mammalian cells. To enable siRNA to be used as an antiviral agent, the nucleic acid delivery barrier must be overcome. Effective local delivery of siRNA to lung tissues is required to reduce the therapeutic dose and minimize systemic adverse effects. To develop a formulation suited for clinical application, complexes of pH-responsive peptides, containing either histidine or 2,3-diaminopropionic acid (Dap), and siRNA were prepared into dry powders by spray drying with mannitol, which was used as a bulking agent. The spray-dried (SD) powders were characterized and found to be suitable for inhalation with good stability, preserving the integrity of the siRNA as well as the biological and antiviral activities. The formulations mediated highly effective in vitro delivery of antiviral siRNA into mammalian lung epithelial cells, leading to significant inhibition of viral replication when the transfected cells were subsequently challenged with H1N1 influenza virus. SD siRNA powders containing pH-responsive peptides are a promising inhalable formulation to deliver antiviral siRNA against influenza and are readily adapted for the treatment of other respiratory diseases.

Co-delivery of PD-L1- and EGFR-targeting siRNAs by synthetic PEG12-KL4 peptide to the lungs as potential strategy against non-small cell lung cancer.

BACKGROUND: Small interfering RNA (siRNA) holds great promise for treating various lung diseases, but the lack of safe and efficient pulmonary siRNA delivery systems has hindered its advance into the clinics. The epidermal growth factor receptor (EGFR) which promotes cell proliferation, and the programmed cell death ligand 1 (PD-L1) which plays a crucial role in suppressing cytotoxic T cells activity, are two important targets for treating non-small cell lung cancer (NSCLC). Here, we explored the potential of PEG12-KL4, a synthetic peptide, to deliver siRNA to various NSCLC cells and to lung tissues in mice. METHODS: PEG12-KL4 was used to transfect siRNAs targeted at both EGFR and PD-L1 into NSCLC cells. Immunoblotting was used to evaluate the siRNA silencing effects in HCC827 and NCI-H1975 NSCLC cells. CD8+ T cell-mediated NSCLC cell killing was employed to demonstrate the functional effects of PD-L1 siRNA knock-down. Fluorescent siRNAs were used to visualise siRNA uptake in cells as well as to enable biodistribution studies in BALB/c mice. RESULTS: Our results showed that PEG12-KL4 was efficient in mediating siRNA knock-down of EGFR and PD-L1 in various NSCLC cells. Importantly, the PEG12-KL4 peptide enabled significantly better siRNA delivery than the commercial Lipofectamine 2000 reagent. We hypothesised that PEG12-KL4 peptide enabled siRNA to either escape from or bypass endosomal degradation as indicated by confocal fluorescence imaging. Notably, combined knock-down of EGFR and PD-L1 in NCI-H1975 cells resulted in better effector T cell-mediated cancer cell killing than knock-down of PD-L1 alone. Moreover, biodistribution of PEG12-KL4/siRNA complexes following intravenous administration revealed poor lung delivery with the fluorescent siRNA accumulating in the liver. In contrast, intratracheal delivery of PEG12-KL4/siRNA complexes resulted in the fluorescent siRNA to be detected in the lung with retarded renal excretion. CONCLUSION: In conclusion, we demonstrated that the co-delivery of siRNAs targeting EGFR and PD-L1 using PEG12-KL4 is feasible and represents a promising future strategy to treat NSCLC, whereby pulmonary siRNA delivery is favourable to intravenous administration.

Oral Delivery of Photopolymerizable Nanogels Loaded with Gemcitabine for Pancreatic Cancer Therapy: Formulation Design, and in vitro and in vivo Evaluations.

Background: Gemcitabine (GEM) faces challenges of poor oral bioavailability and extensive first-pass metabolism. Currently, only injectable formulations are available for clinical use. Hence, there is an urgent demand for the development of advanced, efficacious, and user-friendly dosage forms to maintain its status as the primary treatment for pancreatic ductal adenocarcinoma (PDAC). Nanogels (NGs) offer a novel oral drug delivery system, ideal for hydrophilic compounds like GEM. This study aims to develop NGs tailored for GEM delivery, with the goal of enhancing cellular uptake and gastrointestinal permeability for improved administration in PDAC patients. Methods: We developed cross-linked NGs via photopolymerization of methacryloyl for drug delivery of GEM. We reveal characterization, cytotoxicity, and cellular uptake studies in Caco-2 and MIA PaCa-2 cells. In addition, studies of in vitro permeability and pharmacokinetics were carried out to evaluate the bioavailability of the drug. Results: Our results show NGs, formed via photopolymerization of methacryloyl, had a spherical shape with a size of 233.91±7.75 nm. Gemcitabine-loaded NGs (NGs-GEM) with 5% GelMA exhibited efficient drug loading (particle size: 244.07±19.52 nm). In vitro drug release from NGs-GEM was slower at pH 1.2 than pH 6.8. Cellular uptake studies indicated significantly enhanced uptake in both MIA PaCa-2 and Caco-2 cells. While there was no significant difference in GEM's AUC and Cmax between NGs-GEM and free-GEM groups, NGs-GEM showed markedly lower dFdU content (10.07 hr∙µg/mL) compared to oral free-GEM (19.04 hr∙µg/mL) after oral administration (p<0.01), highlighting NGs' efficacy in impeding rapid drug metabolism and enhancing retention. Conclusion: In summary, NGs enhance cellular uptake, inhibit rapid metabolic degradation of GEM, and prolong retention after oral administration. These findings suggest NGs-GEM as a promising candidate for clinical use in oral pancreatic cancer therapy.

Synergistic combination of antimicrobial peptide and isoniazid as inhalable dry powder formulation against multi-drug resistant tuberculosis.

Multidrug-resistant tuberculosis (MDR-TB) has posed a serious threat to global public health, and antimicrobial peptides (AMPs) have emerged to be promising candidates to tackle this deadly infectious disease. Previous study has suggested that two AMPs, namely D-LAK120-A and D-LAK120-HP13, can potentiate the effect of isoniazid (INH) against mycobacteria. In this study, the strategy of combining INH and D-LAK peptide as a dry powder formulation for inhalation was explored. The antibacterial effect of INH and D-LAK combination was first evaluated on three MDR clinical isolates of Mycobacteria tuberculosis (Mtb). The minimum inhibitory concentrations (MICs) and fractional inhibitory concentration indexes (FICIs) were determined. The combination was synergistic against Mtb with FICIs ranged from 0.25 to 0.38. The INH and D-LAK peptide at 2:1 mole ratio (equivalent to 1: 10 mass ratio) was identified to be optimal. This ratio was adopted for the preparation of dry powder formulation for pulmonary delivery, with mannitol used as bulking excipient. Spherical particles with mass median aerodynamic diameter (MMAD) of around 5 µm were produced by spray drying. The aerosol performance of the spray dried powder was moderate, as evaluated by the Next Generation Impactor (NGI), with emitted fraction and fine particle fraction of above 70 % and 45 %, respectively. The circular dichroism spectra revealed that both D-LAK peptides retained their secondary structure after spray drying, and the antibacterial effect of the combination against the MDR Mtb clinical isolates was successfully preserved. The combination was found to be effective against MDR Mtb isolates with KatG or InhA mutations. Overall, the synergistic combination of INH with D-LAK peptide formulated as inhaled dry powder offers a new therapeutic approach against MDR-TB.

Conformational flexibility determines selectivity and antibacterial, antiplasmodial, and anticancer potency of cationic α-helical peptides.

We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.

Delivery technology of inhaled therapy for asthma and COPD.

Inhaled therapy is the cornerstone of the management of asthma and chronic obstructive pulmonary disease (COPD). Drugs such as bronchodilators and corticosteroids are administered directly to the airways for local effect and rapid onset of action while systemic exposure and side effects are minimized. There are four major types of inhaler devices used clinically to generate aerosols for inhalation, namely, pressurized metered-dose inhalers (pMDIs), nebulizers, Soft Mist™ inhalers (SMIs) and dry powder inhalers (DPIs). Each of them has its own unique characteristics that can target different patient groups. For instance, patients' inhaler technique is critical for pMDIs and SMIs to achieve proper drug deposition in the lung, which could be challenging for some patients. Nebulizers are designed to deliver aerosols to patients during tidal breathing, but they require electricity to operate and are less portable than other devices. DPIs are the only device that delivers aerosols in dry powder form with better stability, but they rely on patients' inspiration effort for powder dispersion, rendering them unsuitable for patients with compromised lung function. Choosing a device that can cater for the need of individual patient is paramount for effective inhaled therapy. This chapter provides an overview of inhaled therapy for the management of asthma and COPD. The operation principles, merits and limitations of different delivery technologies are examined. Looking ahead, the challenges of delivering novel therapeutics such as biologics through the pulmonary route are also discussed.

Inhalable neutralizing antibodies - promising approach to combating respiratory viral infections.

Monoclonal antibodies represent an exciting class of therapeutics against respiratory viral infections. Notwithstanding their specificity and affinity, the conventional parenteral administration is suboptimal in delivering antibodies for neutralizing activity in the airways due to the poor distribution of macromolecules to the respiratory tract. Inhaled therapy is a promising approach to overcome this hurdle in a noninvasive manner, while advances in antibody engineering have led to the development of unique antibody formats which exhibit properties desirable for inhalation. In this Opinion, we examine the major challenges surrounding the development of inhaled antibodies, identify knowledge gaps that need to be addressed and provide strategies from a drug delivery perspective to enhance the efficacy and safety of neutralizing antibodies against respiratory viral infections.

Enhanced powder dispersion of dual-excipient spray-dried powder formulations of a monoclonal antibody and its fragment for local treatment of severe asthma.

The advent of biologics has brought renewed hope for patients with severe asthma, a condition notorious for being hampered by poor response to conventional therapies and adverse drug reactions owing to corticosteroid dependence. However, biologics are administered as injections, thereby precluding the benefits inhalation therapy could offer such as increased bioavailability at the site of action, minimal systemic side effects, non-invasiveness, and self-administration. Here, 2-hydroxypropyl-beta-cyclodextrin and ʟ-leucine were co-spray-dried, as protein stabiliser and dispersion enhancer, respectively, at various weight ratios to produce a series of formulation platforms. Powder aerosolisation characteristics and particle morphology were assessed for suitability for pulmonary delivery. The selected platform with the best aerosol performance, a 1:1 ratio of the excipients, was then incorporated with a monoclonal antibody directed against IL-4 receptor alpha or its antigen-binding fragment. The dual-excipient antibody formulations exhibited emitted fraction of at least 80% and fine particle fraction exceeding 60% in cascade impactor study, while the residual moisture content was within a desirable range between 1% and 3%. The in vitro antigen-binding ability and inhibitory potency of the spray-dried antibody were satisfactorily preserved. The results from this study corroborate the viability of inhaled solid-state biomacromolecules as a promising treatment approach for asthma.

Synthesis and Properties of Sucrose- and Lactose-Based Aromatic Ester Surfactants as Potential Drugs Permeability Enhancers.

The delivery of therapeutics across biological membranes (e.g., mucosal barriers) by avoiding invasive routes (e.g., injection) remains a challenge in the pharmaceutical field. As such, there is the need to discover new compounds that act as drug permeability enhancers with a favorable toxicological profile. A valid alternative is represented by the class of sugar-based ester surfactants. In this study, sucrose and lactose alkyl aromatic and aromatic ester derivatives have been synthesized with the aim to characterize them in terms of their physicochemical properties, structure-property relationship, and cytotoxicity, and to test their ability as permeability enhancer agents across Calu-3 cells. All of the tested surfactants showed no remarkable cytotoxic effect on Calu-3 cells when applied both below and above their critical micelle concentration. Among the explored molecules, lactose p-biphenyl benzoate (URB1420) and sucrose p-phenyl benzoate (URB1481) cause a reversible ~30% decrease in transepithelial electrical resistance (TEER) with the respect to the basal value. The obtained result matches with the increased in vitro permeability coefficients (Papp) calculated for FTIC-dextran across Calu-3 cells in the presence of 4 mM solutions of these surfactants. Overall, this study proposes sucrose- and lactose-based alkyl aromatic and aromatic ester surfactants as novel potential and safe permeation enhancers for pharmaceutical applications.

Neutralisation of SARS-CoV-2 by monoclonal antibody through dual targeting powder formulation.

Neutralising monoclonal antibody (mAb) is an important weapon in our arsenal for combating respiratory viral infections. However, the effectiveness of neutralising mAb has been impeded by the rapid emergence of mutant variants. Early administration of broad-spectrum mAb with improved delivery efficiency can potentially enhance efficacy and patient outcomes. WKS13 is a humanised mAb which was previously demonstrated to exhibit broad-spectrum activity against SARS-CoV-2 variants. In this study, a dual targeting formulation strategy was designed to deliver WKS13 to both the nasal cavity and lower airways, the two critical sites of infection caused by SARS-CoV-2. Dry powders of WKS13 were first prepared by spray drying, with cyclodextrin used as stabiliser excipient. Two-fluid nozzle (TFN) was used to produce particles below 5 µm for lung deposition (C-TFN formulation) and ultrasonic nozzle (USN) was used to produce particles above 10 µm for nasal deposition (C-USN formulation). Gel electrophoresis and size exclusion chromatography studies showed that the structural integrity of mAb was successfully preserved with no sign of aggregation after spray drying. To achieve dual targeting property, C-TFN and C-USN were mixed at various ratios. The aerosolisation property of the mixed formulations dispersed from a nasal powder device was examined using a Next Generation Impactor (NGI) coupled with a glass expansion chamber. When the ratio of C-TFN in the mixed formulation increased, the fraction of particles deposited in the lung increased proportionally while the fraction of particles deposited in the nasal cavity decreased correspondingly. A customisable aerosol deposition profile could therefore be achieved by manipulating the mixing ratio between C-TFN and C-USN. Dual administration of C-TFN and C-USN powders to the lung and nasal cavity of hamsters, respectively, was effective in offering prophylactic protection against SARS-CoV-2 Delta variant. Viral loads in both the lung tissues and nasal wash were significantly reduced, and the efficacy was comparable to systemic administration of unformulated WKS13. Overall, dual targeting powder formulation of neutralising mAb is a promising approach for prophylaxis of respiratory viral infections. The ease and non-invasive administration of dual targeting nasal powder may facilitate the widespread distribution of neutralising mAb during the early stage of unpredictable outbreaks.

Bolaamphiphile Analogues of 12-bis-THA Cl2 Are Potent Antimicrobial Therapeutics with Distinct Mechanisms of Action against Bacterial, Mycobacterial, and Fungal Pathogens.

12-Bis-THA Cl2 [12,12'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications. IMPORTANCE While we face an acute threat from antibiotic resistant bacteria and a lack of new classes of antibiotic, there are many effective antimicrobials which have limited application due to concerns regarding their toxicity and which could be more useful if such risks are reduced or eliminated. We modified a bolalipid antiseptic used in throat lozenges to see if it could be made more effective against some of the highest-priority bacteria and less toxic. We found that structural modifications that rendered the lipid more toxic against human cells made it less toxic in infection models and we could effectively treat caterpillars infected with either Mycobacterium tuberculosis, methicillin resistant Staphylococcus aureus, or Acinetobacter baumannii. The study provides a rationale for further adaptation toward diversifying the range of indications in which this class of antimicrobial may be used.