Investigating the suitability of high content image analysis as a tool to assess the reversibility of foamy alveolar macrophage phenotypes in vitro.

Many potential inhaled medicines fail during development due to the induction of a highly vacuolated or “foamy” alveolar macrophage phenotype response in pre-clinical studies. There is limited understanding if this response to an inhaled stimulus is adverse or adaptive, and additionally if it is a transient or irreversible process. The aim of this study was to evaluate whether high content image analysis could distinguish between different drug-induced foamy macrophage phenotypes and to determine the extent of the reversibility of the foamy phenotypes by assessing morphological changes over time. Alveolar-like macrophages derived from the human monocyte cell line U937 were exposed for 24 h to compounds known to induce a foamy macrophage phenotype (amiodarone, staurosporine) and control compounds that are not known to cause a foamy macrophage phenotype in vitro (fluticasone and salbutamol). Following drug stimulation, the cells were rested in drug-free media for the subsequent 24 or 48 h. Cell morphometric parameters (cellular and nuclear area, vacuoles numbers and size) and phospholipid content were determined using high content image analysis. The foamy macrophage recovery was dependent on the mechanism of action of the inducer compound. Amiodarone toxicity was associated with phospholipid accumulation and morphometric changes were reversed when the stimulus was removed from culture environment. Conversely cells were unable to recover from exposure to staurosporine which initiates the apoptosis pathway. This study shows that high content analysis can discriminate between different phenotypes of foamy macrophages and may contribute to better decision making in the process of new drug development.Peer reviewedFinal Published versio

Physicochemical characterisation of inhalation grade lactose after the removal of intrinsic fines

Lactose is a common excipient in Dry Powder Inhaler (DPI) formulations, used as a carrier for the micronized drug particles. The presence of intrinsic lactose fines in the formulation influences its performance and their role and interactions between the lactose carrier and the micronized drug is still not fully understood. As a first step towards this investigation, “clean” lactose, with removed fines, was produced via wet decantation. Ethanol and isopropyl alcohol have been used in wet decantation, successfully removing lactose fines from the surface of the coarse particles. Differential Scanning Calorimetry (DSC) was employed to show that the powders maintained their crystalline character. Scanning Electron Microscopy (SEM) showed tomahawk-shaped particles in all the powders and some surface alteration occurring after decantation. An airflow titration method using laser diffraction (LDA) allowed the estimation of the removal of fines as well as the particle size distributions, while the non-polar and the polar components of the surface energy of the powders were calculated via Inverse Gas Chromatography-Surface Energy Analysis (iGC-SEA). As both solvents successfully removed fines, we propose the addition of isopropyl alcohol in the list of organic solvents suitable for this purpose.Peer reviewe

‘Great ease and simplicity of action’: Dr Nelson’s Inhaler and the origins of modern inhalation therapy

This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.Unveiled at the conclusion of a meeting of the Royal Medical and Chirurgical Society in 1861,[1] ‘Dr Nelson’s Improved Inhaler’ was one of the most important milestones in the genesis of reliable treatment of respiratory ailments in the modern era. Affordable and suitable for self-medication, the Dr Nelson’s Inhaler offered simple and reliable relief for patients with respiratory and pulmonary ailments. Conspicuous for its modesty and simplicity, it was one of the most widely produced, reproduced, and used inhalation devices in the final third of the nineteenth century. By reconstructing the ‘biography’ of the Nelson Inhaler, this article will attempt to sketch a network of medical and commercial interests and expertise in London which aligned in the 1860s to help establish inhalation as a popular, inexpensive, and trusted form of medical therapy for pulmonary ailments. This article will look at what connects physicians, apothecaries, and patients in the era: the medicines and technologies that were prescribed, made, bought, and which caused wellness, side-effects, and even death. This approach allows us to develop a narrative of respiratory illness as it was experienced by practitioners and patients alike.Peer reviewe

Optimizing the Entrainment Geometry of a Dry Powder Inhaler: Methodology and Preliminary Results.

This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s11095-016-1992-3PURPOSE: For passive dry powder inhalers (DPIs) entrainment and emission of the aerosolized drug dose depends strongly on device geometry and the patient's inhalation manoeuvre. We propose a computational method for optimizing the entrainment part of a DPI. The approach assumes that the pulmonary delivery location of aerosol can be determined by the timing of dose emission into the tidal airstream. METHODS: An optimization algorithm was used to iteratively perform computational fluid dynamic (CFD) simulations of the drug emission of a DPI. The algorithm seeks to improve performance by changing the device geometry. Objectives were to achieve drug emission that was: A) independent of inhalation manoeuvre; B) similar to a target profile. The simulations used complete inhalation flow-rate profiles generated dependent on the device resistance. The CFD solver was OpenFOAM with drug/air flow simulated by the Eulerian-Eulerian method. RESULTS: To demonstrate the method, a 2D geometry was optimized for inhalation independence (comparing two breath profiles) and an early-bolus delivery. Entrainment was both shear-driven and gas-assisted. Optimization for a delay in the bolus delivery was not possible with the chosen geometry. CONCLUSIONS: Computational optimization of a DPI geometry for most similar drug delivery has been accomplished for an example entrainment geometry.Engineering and Physical Sciences Research Council PhD studentship (Grant ID: EP/ M506485/1

Wurster fluidised bed coating of microparticles: Towards scalable production of oral sustained-release liquid medicines for patients with swallowing difficulties

© 2019 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Suspension of microparticles in an easy-to-swallow liquid is one approach to develop sustained-release formulations for children and patients with swallowing difficulties. However, to date production of sustained-release microparticles at the industrial scale has proven to be challenging. The aim of this investigation was to develop an innovative concept in coating sustained-release microparticles using industrial scalable Wurster fluidised bed to produce oral liquid suspensions. Microcrystalline cellulose cores (particle size < 150 µm) were coated with Eudragit® NM 30 D and Eudragit® RS/RL 30 D aqueous dispersions using a fluidised bed coater. A novel approach of periodic addition of a small quantity (0.1% w/w) of dry powder glidant, magnesium stearate, to the coating chamber via an external port was applied throughout the coating process. This method significantly increased coating production yield from less than 50% to up to 99% compared to conventional coating process without the dry powder glidant. Powder rheology tests showed that dry powder glidants increased the tapped density and decreased the cohesive index of coated microparticles. Reproducible microencapsulation of a highly water-soluble drug, metoprolol succinate, was achieved, yielding coated microparticles less than 200 µm in size with 20-hour sustained drug release, suitable for use in liquid suspensions. The robust, scalable technology presented in this study offers an important solution to the long-standing challenges of formulating sustained-release dosage forms suitable for children and older people with swallowing difficulties.Peer reviewedFinal Published versio

Development of a New Dislodgeable Foliar Residue Analytical Laboratory Method for Pesticides

© 2022 The Author(s) . Published by Oxford University Press on behalf of the British Occupational Hygiene Society. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/The dislodgeable foliar residue (DFR) is the amount of pesticide that exists on foliage after the pesticide has dried and which could dislodge to the skin or clothes of workers and is a key parameter for non-dietary risk assessments required to demonstrate safe use for pesticide registration. DFR data in the literature are described as insufficiently reliable, limited, and encompasses considerable statistical uncertainties. The purpose of this article is to describe a newly developed laboratory method for the quantification of DFR with an illustrative example. The laboratory method reflected available field DFR methodology but involved controlled application of droplets to leaves and validation of the wash-off process used to remove the residue from the leaf surface before the analytical quantification. A very high level of accuracy (99.7-102.1%) and precision (±1.5%) was achieved. Residue data generated from the illustrated application of the method showed a robust normal distribution, unlike field studies. The method is deemed to be controllable, cost-efficient, and time-saving, taking hours rather than days. This enables the generation of more data to allow extrapolation between the generated data by investigating multiple factors that may influence DFR. An improved understanding of DFR could save time, money, and resources.Peer reviewe

High Content Image Analysis as a Tool to Morphologically Distinguish Macrophage Activation and Determine Its Importance for Foamy Alveolar Macrophage Responses

Introduction: Lung diseases are an increasing global health burden affecting millions of people worldwide. Only a few new inhaled medicines have reached the market in the last 30 years, in part due to foamy alveolar macrophage (FAM) responses observed in pre-clinical rat studies. The induction mechanism and signaling pathways involved in the development of highly vacuolated ‘foamy’ phenotype is not known. Furthermore, it has not been determined if these observations are adaptive or adverse responses. Aim: To determine if high content image analysis techniques can distinguish between alveolar macrophage activation (LPS/IFN-γ activated and IL-4 activated macrophages) and if this could be applied to understanding the generation of ‘foamy’ macrophage phenotypes. Methods: NR8383 rat alveolar macrophages were stimulated with a mix of cytokines (LPS/IFN-γ or IL-4) for 24 h. The cells were further exposed to FAM inducing-compounds amiodarone and staurosporine. Following 24 h incubation, phagocytosis and lipid accumulation were measured using flow cytometry and high content image analysis techniques. The alveolar macrophages responses after exposure to cytokines were assessed by evaluation: (i) cell surface and biochemical markers such as: nitric oxide production, arginase-1 activity and MRC-1 receptor expression (ii) cellular morphology (iii) cellular functionality (phagocytic activity and lipids accumulation). Results: Macrophages activated with LPS/IFN-γ showed distinct morphological (increased vacuolation) features and functionality (increased lipidosis, decreased phagocytic activity). Foamy macrophage phenotypes induced by amiodarone also displayed characteristics of proinflammatory macrophages (significantly increased nitric oxide production, increased vacuolation and lipidosis and decreased phagocytosis). In contrast, staurosporine treatment resulted in increased NO production, as well as arginase-1 activity. Conclusion: High content image analysis was able to determine distinct differences in morphology between non-activated and LPS/IFN-γ activated macrophages, characterized by increased vacuolation and lipidosis. When exposed to compounds that induce a FAM phenotype, healthy non-activated macrophages displayed proinflammatory (amiodarone) or pro-apoptotic (staurosporine) characteristics but these responses were independent of a change in activation status. This technique could be applied in early drug discovery safety assessment to identify immune responses earlier and increase the understanding of alveolar macrophage responses to new molecules challenge in development of new inhalation therapies, which in turn will enhance decision-making in an early safety assessment of novel drug candidates.Peer reviewedFinal Published versio

Establishing the importance of oil-membrane interactions on the transmembrane diffusion of physicochemically diverse compounds

© 2016. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This is a pre-copyedited, author produced PDF of an article accepted for publication in International Journal of Phrmaceutics following peer review. The final, definitive version of this paper Vol 56 (1-2): 429-437 , first published online March 21, 2016, is available on line at doi: 10.1016/j.ijpharm.2016.03.020The diffusion process through a non-porous barrier membrane depends on the properties of the drug, vehicle and membrane. The aim of the current study was to investigate whether a series of oily vehicles might have the potential to interact to varying degrees with synthetic membranes and to determine whether any such interaction might affect the permeation of co-formulated permeants: methylparaben (MP); butylparaben (BP) or caffeine (CF). The oils (isopropyl myristate (IPM), isohexadecane (IHD), hexadecane (HD), oleic acid (OA) and liquid paraffin (LP)) and membranes (silicone, high density polyethylene and polyurethane) employed in the study were selected such that they displayed a range of different structural, and physicochemical properties. Diffusion studies showed that many of the vehicles were not inert and did interact with the membranes resulting in a modification of the permeants’ flux when corrected for membrane thickness (e.g. normalized flux of MP increased from 1.25 ± 0.13 μg cm−1 h−1 in LP to 17.94 ± 0.25 μg cm−1 h−1in IPM). The oils were sorbed differently to membranes (range of weight gain: 2.2 ± 0.2% for polyurethane with LP to 105.6 ± 1.1% for silicone with IHD). Membrane interaction was apparently dependent upon the physicochemical properties including; size, shape, flexibility and the Hansen solubility parameter values of both the membranes and oils. Sorbed oils resulted in modified permeant diffusion through the membranes. No simple correlation was found to exist between the Hansen solubility parameters of the oils or swelling of the membrane and the normalized fluxes of the three compounds investigated. More sophisticated modelling would appear to be required to delineate and quantify the key molecular parameters of membrane, permeant and vehicle compatibility and their interactions of relevance to membrane permeation.Peer reviewe

Water Uptake by Evaporating pMDI Aerosol Prior to Inhalation Affects Both Regional and Total Deposition in the Respiratory System

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)As pulmonary drug deposition is a function of aerosol particle size distribution, it is critical that the dynamics of particle formation and maturation in pMDI sprays in the interim between generation and inhalation are fully understood. This paper presents an approach to measure the evaporative and condensational fluxes of volatile components and water from and to solution pMDI droplets following generation using a novel technique referred to as the Single Particle Electrodynamic Lung (SPEL). In doing so, evaporating aerosol droplets are shown capable of acting as condensation nuclei for water. Indeed, we show that the rapid vaporisation of volatile components from a volatile droplet is directly correlated to the volume of water taken up by condensation. Furthermore, a significant volume of water is shown to condense on droplets of a model pMDI formulation (hydrofluoroalkane (HFA), ethanol and glycerol) during evaporative droplet ageing, displaying a dramatic shift from a core composition of a volatile species to that of predominantly water (non-volatile glycerol remained in this case). This yields a droplet with a water activity of 0.98 at the instance of inhalation. The implications of these results on regional and total pulmonary drug deposition are explored using the International Commission of Radiological Protection (ICRP) deposition model, with an integrated semi-analytical treatment of hygroscopic growth. Through this, droplets with water activity of 0.98 upon inhalation are shown to produce markedly different dose deposition profiles to those with lower water activities at the point of inspiration.Peer reviewe

Polymer architecture dictates thermoreversible gelation in engineered emulsions stabilised with branched copolymer surfactants

© The Royal Society of Chemistry 2022. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.The generation of materials that switch from a liquid to gel state upon warming can enable new healthcare technologies with improved functionality, such as in situ gel-forming materials for drug delivery to topical or parenteral sites. The majority of these materials are aqueous polymer solutions, which then suffer from an inability to solubilise hydrophobic drugs. This study investigates the generation of thermoresponsive “engineered emulsions” which are low-viscosity emulsions at low temperature and switch to a gel state upon warming. This is achieved by the synthesis of novel branched copolymer surfactants (BCS) containing di(ethylene glycol) methyl ether methacrylate (DEGMA) as a thermoresponsive component giving a lower critical solution temperature (LCST). The copolymers were employed as emulsifiers to prepare 1 : 1 dodecane:water emulsion systems. The effect of polymer architecture is shown to be intimately linked to the rheology of these systems, where branching, elevation of molecular weight, and the presence of hydrophobic end groups is demonstrated to be commensurate with gel formation upon heating. Mechanisms of gel formation were probed by small-angle neutron scattering, which demonstrated that the branched copolymer surfactants formed oblate ellipsoids in solution that grew anisotropically with temperature, forming larger disk-like nanoparticles. The formation of these elongated particles leads to thickening of the emulsions, whilst connectivity of the aggregates and BCS at the oil–water interface is required for gel formation to occur. Overall, the study provides design principles for this novel class of thermoresponsive material with great potential in healthcare, cosmetic, and energy applications.Peer reviewe