Search

OBJECTIVES: A human nasal epithelial mucosa (NEM) on-a-chip is developed integrated with a novel carbon nanofibers-modified carbon electrode for real-time quantitative monitoring of in vitro nasal drug delivery. The integration of platinum electrodes in the chip also enables real-time measurement of transepithelial electrical resistance (TEER). METHODS: The air-liquid interface culture of nasal epithelial RPMI 2650 cells in the NEM-on-a-chip was optimized to mimic the key functional characteristics of the human nasal mucosa. The epithelial transport of ibuprofen in the NEM-on-a-chip was electrochemically monitored in real-time under static and physiologically realistic dynamic flow conditions. RESULTS: The NEM-on-a-chip mimics the mucus production and nasal epithelial barrier function of the human nasal mucosa. The real-time drug quantification by the NEM-on-a-chip was validated versus the high-performance liquid chromatography method. The drug transport rate monitored in the NEM-on-a-chip was influenced by the flow in the bottom compartment of the chip, highlighting the importance of emulating the dynamic in vivo condition for nasal drug transport studies. CONCLUSION: This novel NEM-on-a-chip can be a low-cost and time-efficient alternative to the costly laborious conventional techniques for in vitro nasal drug transport assays. Importantly, its dynamic microenvironment enables conducting nasal drug transport tests under physiologically relevant dynamic conditions.

Current cystic fibrosis (CF) treatment strategies are primarily focused on oral/inhaled anti-inflammatories and antibiotics, resulting in a considerable treatment burden for CF patients. Therefore, combination treatments consisting of anti-inflammatories with antibiotics could reduce the CF treatment burden. However, there is an imperative need to understand the potential drug-drug interactions of these combination treatments to determine their efficacy. Thus, this study aimed to determine the interactions of the anti-inflammatory agent Ibuprofen with each of the CF-approved inhaled antibiotics (Tobramycin, Colistin and its prodrug colistimethate sodium/Tadim) and anti-bacterial and anti-inflammatory efficacy. Chemical interactions of the Ibuprofen:antibiotic combinations were elucidated using High-Resolution Mass-Spectrometry (HRMS) and 1H NMR. HRMS showed pairing of Ibuprofen and Tobramycin, further confirmed by 1H NMR whilst no pairing was observed for either Ibuprofen:Colistin or Ibuprofen:Tadim combinations. The anti-bacterial activity of the combinations against Pseudomonas aeruginosa showed that neither paired nor non-paired Ibuprofen:antibiotic therapies altered the anti-bacterial activity. The anti-inflammatory efficacy of the combination therapies was next determined at two different concentrations (Low and High) using in vitro models of NuLi-1 (healthy) and CuFi-1 (CF) cell lines. Differential response in the anti-inflammatory efficacy of Ibuprofen:Tobramycin combination was observed between the two concentrations due to changes in the structural conformation of the paired Ibuprofen:Tobramycin complex at High concentration, confirmed by 1H NMR. In contrast, the non-pairing of the Ibuprofen:Colistin and Ibuprofen:Tadim combinations showed a significant decrease in IL-8 secretion at both the concentrations. Importantly, all antibiotics alone showed anti-inflammatory properties, highlighting the inherent anti-inflammatory properties of these antibiotics.

Background: Lymphangioleiomyomatosis (LAM) is characterized by growth of smooth muscle-like cells in the lungs that spread to other organs via lymphatic vessels. Current oral rapamycin treatment is limited by low bioavailability of approximately 15%. Aim: The effect of inhaled rapamycin solid lipid nanoparticles (Rapa-SLNs) size on its penetration through the lymphatics. Method: Three Rapa-SLN formulations (200-1000 nm) were produced and assessed for particle characteristics and further for toxicity and performance in vitro. Results: Rapa-SLNs of 200 nm inhibited proliferation in TSC2-negative mouse embryonic fibroblast cells and penetrated the respiratory epithelium and lymphatic endothelium significantly faster compared with free rapamycin and larger Rapa-SLNs. Conclusion: Rapa-SLN approximately 200 nm allows efficient entry of rapamycin into the lymphatic system and is therefore a promising treatment for LAM patients.

For the past 50 years, the route of inhalation has been utilized to administer therapies to treat a variety of respiratory and pulmonary diseases. When compared with other drug administration routes, inhalation offers a targeted, non-invasive approach to deliver rapid onset of drug action to the lung, minimizing systemic drug exposure and subsequent side effects. However, despite advances in inhaled therapies, there is still a need to improve the preclinical screening and the efficacy of inhaled therapeutics. Innovative in vitro models of respiratory physiology to determine therapeutic efficacy of inhaled compounds have included the use of organoids, micro-engineered lung-on-chip systems and sophisticated bench-top platforms to enable a better understanding of pulmonary mechanisms at the molecular level, rapidly progressing inhaled therapeutic candidates to the clinic. Furthermore, the integration of complementary ex vivo models, such as precision-cut lung slices (PCLS) and isolated perfused lung platforms have further advanced preclinical drug screening approaches by providing in vivo relevance. In this review, we address the challenges and advances of in vitro models and discuss the implementation of ex vivo inhaled drug screening models. Specifically, we address the importance of understanding human in vivo pulmonary mechanisms in assessing strategies of the preclinical screening of drug efficacy, toxicity and delivery of inhaled therapeutics.

Fluticasone propionate uptake in the presence of a proprietary cell-penetrating peptide (human stimulus factor, [HSF]) based on the N-terminal domain of lactoferrin was studied, alone and in combination with salmeterol, using an air interface Calu-3 epithelial model. The HSF enhanced uptake and transport of fluticasone propionate across the epithelial barrier when alone and in presence of salmeterol. This was attributed to transcellular-mediated uptake. This HSF is a promising peptide for delivery of therapeutics where enhanced epithelial penetrating is required.

© 2018, American Association of Pharmaceutical Scientists. The purpose of this study was to present a novel and simple drug deposition method to evaluate drug transport of aerosol microparticles across airway epithelial cells. Microparticles containing ciprofloxacin HCl (Cip) and doxycycline (Dox), alone or in a 50:50% w/w ratio, were spray dried and suspended using 2H, 3H-perfluoropentane, model propellant. The suspension was then used to assess deposition, and transport of these drug microparticles across sub-bronchial epithelial Calu-3 cells was also studied. In comparison with other methods of depositing microparticles, this proposed method, using drug suspended in HPFP, provides control over the amount of drugs applied on the surface of the cells. Therefore, cell permeability studies could be conducted with considerably smaller and more reproducible doses, without the physicochemical characteristics of the drugs being compromised or the use of modified pharmacopeia impactors. The suspension of microparticles in HPFP as presented in this study has provided a non-toxic, simple, and reproducible novel method to deliver and study the permeability of specific quantity of drugs across respiratory epithelial cells in vitro.

© 2018 Elsevier B.V. Despite many therapeutic advancements over the past decade, the continued rise in chronic inflammatory lung diseases incidence has driven the need to identify and develop new therapeutic strategies, with superior efficacy to treat these diseases. Statins are one class of drug that could potentially be repurposed as an alternative treatment for chronic lung diseases. They are currently used to treat hypercholesterolemia by inhibiting the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, that catalyses the rate limiting step in the mevalonate biosynthesis pathway, a key intermediate in cholesterol metabolism. Recent research has identified statins to have other protective pleiotropic properties including anti-inflammatory, anti-oxidant, muco-inhibitory effects that may be beneficial for the treatment of chronic inflammatory lung diseases. However, clinical studies have yielded conflicting results. This review will summarise some of the current evidences for statins pleiotropic effects that could be applied for the treatment of chronic inflammatory lung diseases, their mechanisms of actions, and the potential to repurpose statins as an inhaled therapy, including a detailed discussion on their different physical-chemical properties and how these characteristics could ultimately affect treatment efficacies. The repurposing of statins from conventional anti-cholesterol oral therapy to inhaled anti-inflammatory formulation is promising, as it provides direct delivery to the airways, reduced risk of side effects, increased bioavailability and tailored physical-chemical properties for enhanced efficacy.

© 2016 Elsevier B.V. The aim of this study was to incorporate an optimized RPMI2650 nasal cell model into a 3D printed model of the nose to test deposition and permeation of drugs intended for use in the nose. The nasal cell model was optimized for barrier properties in terms of permeation marker and mucus production. RT-qPCR was used to determine the xenobiotic transporter gene expression of RPMI 2650 cells in comparison with primary nasal cells. After 14 days in culture, the cells were shown to produce mucus, and to express TEER (define) values and sodium fluorescein permeability consistent with values reported for excised human nasal mucosa. In addition, good correlation was found between RPMI 2650 and primary nasal cell transporter expression values. The purpose-built 3D printed model of the nose takes the form of an expansion chamber with inserts for cells and an orifice for insertion of a spray drug delivery device. This model was validated against the FDA glass chamber with cascade impactors that is currently approved for studies of nasal products. No differences were found between the two apparatus. The apparatus including the nasal cell model was used to test a commercial nasal product containing budesonide (Rhinocort, AstraZeneca, Australia). Drug deposition and transport studies on RPMI 2650 were successfully performed. The new 3D printed apparatus that incorporates cells can be used as valid in vitro model to test nasal products in conditions that mimic the delivery from nasal devices in real life conditions.

© 2015 American Chemical Society. Biofilm tolerance has become a serious clinical concern in the treatment of nosocomial pneumonia owing to the resistance to various antibiotics. There is an urgent need to develop alternative antimicrobial agents or combination drug therapies that are effective via different mechanisms. Silver nanoparticles (AgNPs) have been developed as an anti-biofilm agent for the treatment of infections associated with the use of mechanical ventilations, such as endotracheal intubation. Meanwhile curcumin, a phenolic plant extract, has displayed natural anti-biofilm properties through the inhibition of bacterial quorum sensing systems. The aim of this study was to investigate the possible synergistic/additive interactions of AgNPs and curcumin nanoparticles (Cur-NPs) against both Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) microorganisms. The combination of AgNPs and Cur-NPs (termed Cur-SNPs) at 100 μg/mL disrupted 50% of established bacterial biofilms (formed on microtiter plates). However, further increase in the concentration of Cur-SNPs failed to effectively eliminate the biofilms. To achieve the same effect, at least 500 μg/mL Cur-NP alone was needed. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) revealed that combination therapy (Cur-SNPs) was the most potent to eradicate preformed biofilm compared to monodrug therapy. These agents are also nontoxic to healthy human bronchial epithelial cells (BEAS2B).

© 2015 American Chemical Society. The aim of this study was to investigate the changes in transport and effectiveness of salbutamol sulfate (SAL) and budesonide (BD) following stimulation with transforming growth factor-β (TGF-β) in mono- and coculture models of bronchial and alveolar epithelium. Primary bronchial and alveolar epithelial cells, grown at air interface on filters, either as monocultures or in coculture with airway smooth muscle cells or alveolar macrophages, respectively, were stimulated with TGF-β. The biological response was modulated by depositing aerosolized SAL and BD on bronchial and alveolar models, respectively. Barrier integrity, permeability to fluorescein-Na, transport of the deposited drug, and the pharmacological response to SAL (cAMP and IL-8 levels) or BD (IL-6 and -8 levels) were measured. While stimulation with TGF-β did not have any significant effect on the transepithelial electrical resistance and permeability to fluorescein-Na in mono- and coculture models, transport of SAL and BD were affected in cultures from some of the patients (6 out of 12 for bronchial and 2 out of 4 for alveolar cells). The bronchial coculture showed a better responsiveness to SAL in terms of cAMP release than the monoculture. In contrast, the difference between alveolar mono- and cocultures to TGF-β mediated interleukin release and its modulation by BD was less pronounced. Our data point to intrinsic differences in the transport of, and responsiveness to, SAL and BD when epithelial cell cultures originate from different patients. Moreover, if the biological responses (e.g., IL-8, cAMP) involve communication between different cell types, coculture models are more relevant to measure such effects than monocultures.

© The Royal Society of Chemistry 2015. The impact of a polyunsaturated fatty acid, arachidonic acid (AA), on membrane fluidity of epithelial cells and subsequent modulation of the drug transport was investigated. Membrane fluidity was assessed using molecular force microscopy. Calu-3 human bronchial epithelial cells were cultured on Transwell® inserts and the cell stiffness was assessed in the absence of fatty acids or in the presence of 30 μM AA. The morphology of the epithelial cells was distinctly different when AA was present, with the cell monolayer becoming more uniform. Furthermore the cell stiffness and variation in stiffness was lower in the presence of AA. In the fat-free medium, the median cell stiffness was 9.1 kPa which dropped to 2.1 kPa following exposure to AA. To further study this, transport of a common β2-agonist, salbutamol sulphate (SS) was measured in the presence of AA and in a fat free medium. The transport of SS was significantly higher when AA was present (0.61 ± 0.09 μg versus 0.11 ± 0.003 μg with and without AA respectively). It was evidenced that AA play a vital role in cell membrane fluidity and drug transport. This finding highlights the significance of the dietary fatty acids in transport and consequentially effectiveness of medications used to treat pulmonary diseases such as asthma. This journal is

Crystalline salbutamol sulphate (SS) is a common β2-agonist used in dry powder inhalers for the treatment of asthma. The solid-state characteristics of SS are import since they govern the stability, and thus efficacy of the drug when incorporated in inhalation medicine. Previous studies have investigated the thermal properties of SS and the complex array of thermal events have been attributed a mixture of melting and/or degradation mechanisms. In order to ascertain the exact thermal transformation processes that SS undergoes, and we utilised a combination of differential scanning calorimetry coupled with quadrupole mass spectrometry and thermogravimetric analysis coupled with Fourier transform infrared spectroscopy over the temperature range 25-500 °C. Based on the coupled thermal analysis data, we proposed that SS undergoes a multi-step degradation mechanism in which the molecule dehydrates loosing water initially, followed by the break up of the secondary amine group and lastly formation of sulphur dioxide. When used in conjunction, the analytical techniques offered significant advantages over the use of thermal analysis alone, offering a better understanding of the transformations occurring to SS following heating.

© 2014 Elsevier Inc. During the process of inhalable formulation development a deep knowledge of the physicochemical characteristics of the drug and formulation components and the biological properties of the airways is necessary. For example, the solubility and lipophilicity of a drug may affect therapeutic efficacy by changing the residence time of the microparticles at the airway surface. Furthermore, the properties of microparticles, such as shape, size and density, as well as the diseases of the respiratory tract, delivery device and inhalation manoeuvre will have an impact on where these microparticles are deposited. The airway epithelium is involved in the pathogenesis and treatment of respiratory diseases. Epithelial cells are directly exposed to the environment and respond to xenobiotics. In some cases, they are the site of action for drug molecules or the drug molecules might need to be transported across the epithelium to arrive at the site of action. The drug particles deposited on the respiratory epithelia have to interact with the mucus lining, dissolve and get transported through this layer. Despite advances in in vitro testing of respiratory epithelial permeability, there is little known about how and where drugs are absorbed at a cellular level and how long they reside in the lung. Therefore, pulmonary permeability assessment of drugs may provide insights that will allow formulations to be developed with optimised therapeutic outcomes. This review focuses on the integration of these physicochemical characteristics with the biological factors to provide a better understanding of the fate of microparticles after deposition on the epithelial cells.

Liposomal ciprofloxacin formulations have been developed with the aim of enhancing lung residence time, thereby reducing the burden of inhaled antimicrobial therapy which requires multiple daily administration due to rapid absorptive clearance of antibiotics from the lungs. However, there is a lack of a predictive methodology available to assess controlled release inhalation delivery systems and their effect on drug disposition. In this study, three ciprofloxacin formulations were evaluated: a liposomal formulation, a solution formulation and a 1:1 combination of the two (mixture formulation). Different methodologies were utilised to study the release profiles of ciprofloxacin from these formulations: (i) membrane diffusion, (ii) air interface Calu-3 cells and (iii) isolated perfused rat lungs. The data from these models were compared to the performance of the formulations in vivo. The solution formulation provided the highest rate of absorptive transport followed by the mixture formulation, with the liposomal formulation providing substantially slower drug release. The rank order of drug release/transport from the different formulations was consistent across the in vitro andex vivo methods, and this was predictive of the profiles in vivo. The use of complimentary in vitro and ex vivo methodologies provided a robust analysis of formulation behaviour, including mechanistic insights, and predicted in vivo pharmacokinetics.© 2013 Elsevier B.V. All rights reserved.

Ciprofloxacin is a well-established broad-spectrum fluoroquinolone antibiotic that penetrates well into the lung tissues; still, the mechanisms of its transepithelial transport are unknown. The contributions of specific transporters, including multidrug efflux transporters, organic cation transporters, and organic anion-transporting polypeptide transporters, to the uptake of ciprofloxacin were investigated in vitro using an air interface bronchial epithelial model. Our results demonstrate that ciprofloxacin is subject to predominantly active influx and a slight efflux component. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

Two solution-based pressurised metered dose inhaler (pMDI) formulations were prepared such that they delivered aerosols with identical mass median aerodynamic diameters, but contained either beclomethasone dipropionate (BDP) alone (glycerol-free formulation) or BDP and glycerol in a 1:1 mass ratio (glycerol-containing formulation). The two formulations were deposited onto Calu-3 respiratory epithelial cell layers cultured at an air interface. Equivalent drug mass (∼1000 ng or ∼2000 ng of the formulation) or equivalent particle number (1000 ng of BDP in the glycerol-containing versus 2000 ng of BDP in the glycerol-free formulation) were deposited as aerosolised particles on the air interfaced surface of the cell layers. The transfer rate of BDP across the cell layer after deposition of the glycerol-free particles was proportional to the mass deposited. In comparison, the transfer of BDP from the glycerol-containing formulation was independent of the mass deposited, suggesting that the release of BDP is modified in the presence of glycerol. The rate of BDP transfer (and the extent of metabolism) over 2 h was faster when delivered in glycerol-free particles, 465.01 ng ± 95.12 ng of the total drug (20.99 ± 4.29%; BDP plus active metabolite) transported across the cell layer, compared to 116.17 ng ± 3.07 ng (6.07 ± 0.16%) when the equivalent mass of BDP was deposited in glycerol-containing particles. These observations suggest that the presence of glycerol in the maturated aerosol particles may influence the disposition of BDP in the lungs.© 2013 Elsevier B.V. All rights reserved.

A series of semi-empirical equations were utilised to design two solution based pressurised metered dose inhaler (pMDI) formulations, with equivalent aerosol performance but different physicochemical properties. Both inhaler formulations contained the drug, beclomethasone dipropionate (BDP), a volatile mixture of ethanol co-solvent and propellant (hydrofluoroalkane-HFA). However, one formulation was designed such that the emitted aerosol particles contained BDP and glycerol, a common inhalation particle modifying excipient, in a 1:1 mass ratio. By modifying the formulation parameters, including actuator orifice, HFA and metering volumes, it was possible to produce two formulations (glycerol-free and glycerol-containing) which had identical mass median aerodynamic diameters (2.4 μm ± 0.1 and 2.5 μm ± 0.2), fine particle dose (≤5 μm; 66 μg ± 6 and 68 μg ± 2) and fine particle fractions (28% ± 2% and 30% ± 1%), respectively. These observations demonstrate that it is possible to engineer formulations that generate aerosol particles with very different compositions to have similar emitted dose and in vitro deposition profiles, thus making them equivalent in terms of aerosol performance. Analysis of the physicochemical properties of each formulation identified significant differences in terms of morphology, thermal properties and drug dissolution of emitted particles. The particles produced from both formulations were amorphous; however, the formulation containing glycerol generated particles with a porous structure, while the glycerol-free formulation generated particles with a primarily spherical morphology. Furthermore, the glycerol-containing particles had a significantly lower dissolution rate (7.8% ± 2.1%, over 180 min) compared to the glycerol-free particles (58.0% ± 2.9%, over 60 min) when measured using a Franz diffusion cell. It is hypothesised that the presence of glycerol in the emitted aerosol particles altered solubility and drug transport, which may have implications for BDP pharmacokinetics after deposition in the respiratory tract.© 2013 Elsevier B.V. All rights reserved.

Background: Chronic obstructive pulmonary disease (COPD) is characterised by mucus hyper-production. This pathology, together with other inflammatory contributions, leads to airway obstruction and breathing complications. Newer therapeutic approaches are of increased interest, including the use of HMG-CoA reductase inhibitors. Retrospective studies have shown that statins are effective in reducing patient mortality and blood cytokines levels. These findings suggest statins may also provide a new therapeutic approach in COPD treatment. Purpose: The aim of the present work was to study the transport of simvastatin (SV) across Calu-3 epithelial cells and to investigate its pharmacological action with respect to reduction in mucus production. Methods: Calu-3 cells were grown under liquid covered culture (LCC) conditions for transport studies in order to demonstrate the ability of SV to transport across the monolayer. For mucus detection, cells were grown under air interface culture (AIC) conditions. Samples collected for microscope analysis were stained with alcian blue; images of the stained cell surface were acquired and the mucus was quantified as the RGBB ratio. Results: SV was transported through the cell monolayer and 'retained' inside the Calu-3 cells. Colour analysis of stained Calu-3 monolayers microscope-images showed that chronic administration of SV for 14 days caused a significant inhibition in mucus production. Conclusion: These findings suggest that local delivery of SV directly to the lungs may provide a promising treatment and potential disease management approach of COPD, with significant effects on mucus reduction. © 2013 Elsevier B.V. All rights reserved.