Mehra Haghi

Time- and passage-dependent characteristics of a Calu-3 respiratory epithelial cell model

Background: Although standard protocols for the study of drug delivery in the upper airways using the sub-bronchial epithelial cell line Calu-3 model, particularly that of the air-liquid interface configuration, are readily available, the model remains un-validated with respect to culture conditions, barrier integrity, mucous secretion, and transporter function. With respect to the latter, the significance of functional P-glycoprotein (P-gp) activity in Calu-3 cells has recently been questioned, despite previous reports demonstrating a significant contribution by the same transporter in limiting drug uptake across the pulmonary epithelium. Therefore, the aim of this study was the standardization of this model as a tool for drug discovery. Methods: Calu-3 cells were grown using air-interfaced condition (AIC) on polyester cell culture supports. Monolayers were evaluated for transepithelial electrical resistance (TEER), permeability to the paracellular marker fluorescein sodium (flu-Na), surface P-gp expression, and functionality. Mucous secretion was also identified by alcian blue staining. Results: TEER and permeability values obtained for Calu-3 monolayers were shown to plateau between day 5 and day 21 in culture with values reaching 474 ± 44 ωcm2 and 2.33 ± 0.36 × 10–7 cm/s, respectively, irrespective of the passage number examined. 32.7 ± 1.49% of Calu-3 cells cultured under these conditions detected positive for cell surface P-gp expression from day 7 onwards. Functional cell surface expression was established by rhodamine 123 drug extrusion assays. Conclusion: This study establishes a clear dependence on culture time and passage number for optimal barrier integrity, mucous secretion, and cell-surface P-gp expression and function in Calu-3 cells. Furthermore it provides initial guidelines for the optimization of this model for high throughput screening applications.

Deposition, diffusion and transport mechanism of dry powder microparticulate salbutamol, at the respiratory epithelia.

The deposition, dissolution and transport of salbutamol base (SB) and salbutamol sulfate (SS) inhalation powders were investigated using the Calu-3 air interface cell culture model and Franz diffusion cell. Drug uptake by cells was studied with respect to deposited dose, drug solubility and hydrophobicity. Furthermore, the role of active transport via organic cationic transporters (OCTs) was studied. SB and SS were processed to have similar diameters (3.09 ± 0.06 μm and 3.07 ± 0.03 μm, respectively) and were crystalline in nature. Analysis of drug wetting, dissolution and diffusion using a conventional in vitro Franz cell (incorporating a cell culture support Transwell polyester membrane) showed diffusion of SB to be slower than that of SS (98.57 ± 4.23 μg after 4 h for SB compared to 98.57 ± 4.01 μg after 15 min for SS). Such observations suggest dissolution to be the rate-limiting step. In comparison, the percentage transfer rate using the air interface Calu-3 cell model suggested SB transport to be significantly faster than SS transport (92.02 ± 4.47 μg of SB compared to 63.76 ± 8.84 μg of SS transported over 4 h), indicating that passive diffusion through the cell plays a role in transport. Furthermore, analysis of SB and SS transport, over a range of deposited doses, suggested the transport rate in the Franz diffusion cell to be limited by wetting of the particle and dissolution into the medium. However, for the cell monolayer, the cell membrane properties regulate the diffusion and transport rate. Analysis of the drug transport in the presence of triethylamine (TEA), a known inhibitor of OCTs, resulted in a significant decrease in drug transport, suggesting an active transport mechanism. The presence of OCTs in this cell line was further validated by Western blot analysis. Finally, the transport of SS from a commercial product (Ventolin Rotacaps) was studied and showed good agreement with the model SS system studied here.

Quercetin solid lipid microparticles: A flavonoid for inhalation lung delivery

Abstract Purpose The aim of the present work was to develop solid lipid microparticles (SLMs), as dry powders containing quercetin for direct administration to the lung. Methods Quercetin microparticles were prepared by o/w emulsification via a phase inversion technique, using tristearin as the lipid component and phosphatidylcholine as an emulsifier. The quercetin SLMs were characterised for morphology, drug loading (15.5%±0.6, which corresponded to an encapsulation efficiency of 71.4%), particle size distribution, response to humidity, crystallinity, thermal behaviour and in vitro respirable fraction. Furthermore, the toxicity and the in vitro transport of the SLMs on an air liquid interface model of the Calu-3 cell line were also investigated using a modified twin-stage impinger apparatus. Results Results showed that quercetin SLMs could be formulated as dry powder suitable for inhalation drug delivery (20.5±3.3% fine particle fraction ⩽4.46μm) that was absorbed, via a linear kinetic model across the Calu-3 monolayer (22.32±1.51% over 4h). In addition, quercetin SLMs were shown to be non-toxic at the concentrations investigated. Interestingly, no apical to basolateral transport of the micronised quercetin was observed over the period of study. Conclusions These observations suggest quercetin diffusion was enhanced by the presence of the lipid/emulsifying excipients in the SLMs; however further studies are necessary to elucidate the exact mechanisms.

Across the pulmonary epithelial barrier: Integration of physicochemical properties and human cell models to study pulmonary drug formulations

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.

Towards the bioequivalence of pressurised metered dose inhalers 2. Aerodynamically equivalent particles (with and without glycerol) exhibit different biopharmaceutical profiles in vitro

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 (∼1000ng or ∼2000ng of the formulation) or equivalent particle number (1000ng of BDP in the glycerol-containing versus 2000ng 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 2h was faster when delivered in glycerol-free particles, 465.01ng±95.12ng of the total drug (20.99±4.29%; BDP plus active metabolite) transported across the cell layer, compared to 116.17ng±3.07ng (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.

Towards the bioequivalence of pressurised metered dose inhalers 1: Design and characterisation of aerodynamically equivalent beclomethasone dipropionate inhalers with and without glycerol as a non-volatile excipient

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 (2.4μm±0.1 and 2.5μm±0.2), fine particle dose (⩽5μm; 66μg±6 and 68μg±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 180min) compared to the glycerol-free particles (58.0%±2.9%, over 60min) 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.

Development of an Inhaled Controlled Release Voriconazole Dry Powder Formulation for the Treatment of Respiratory Fungal Infection

The present research aimed to develop and characterize a sustained release dry powder inhalable formulation of voriconazole (VRZ) for invasive pulmonary aspergillosis. The developed formulations were studied for their in vitro release profile, aerosol, and physicochemical properties as well as interactions with lung epithelia in terms of toxicity and transport/uptake. VRZ and VRZ loaded poly lactide microparticles (VLM) were prepared by aqueous/organic cosolvent and organic spray drying, respectively. Powders were characterized using laser diffraction, differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), dynamic vapor sorption (DVS), and electron microscopy. Aerosol performance was evaluated using an RS01 dry powder inhaler and in vitro cascade impaction. Uptake across Calu-3 lung epithelia was studied, using aerosol deposition of the powder onto cells cultured in an air interface configuration, and compared to dissolution using a conventional dialysis membrane. Additionally, toxicity of VRZ and VLM and the potential impact of transmembrane proteins on uptake were investigated. The particle size and the aerosol performance of spray-dried VRZ and VLM were suitable for inhalation purposes. VRZ exhibited a median volume diameter of 4.52 ± 0.07 μm while VLM exhibited 2.40 ± 0.05 μm. Spray-dried VRZ was crystalline and VLM amorphous as evaluated by DSC and XRPD, and both powders exhibited low moisture sorption between 0 and 90% RH (<1.2% w/w) by DVS. The fine particle fraction (FPF) (% aerosol <5 μm) for the VRZ was 20.86 ± 1.98% while the VLM showed significantly improved performance (p < 0.01) with an FPF of 43.56 ± 0.13%. Both VRZ and VLM were not cytotoxic over a VRZ concentration range of 1.2 nM to 30 μM, and the VLM particles exhibited a sustained release over 48 h after being deposited on the Calu-3 cell line or via conventional dialysis-based dissolution measurements. Lastly, VRZ exhibited polarized transport across epithelia with basal to apical transport being slower than apical to basal. Influx and efflux transports may also play a role as transport was altered in the presence of a number of inhibitors. This study has established an inhalable and sustained release powder of VRZ for targeting invasive pulmonary aspergillosis.

Salbutamol Sulfate Absorption Across Calu-3 Bronchial Epithelia Cell Monolayer is Inhibited in the Presence of Common Anionic NSAIDs

Purpose. The aim of this study was to characterize the permeability kinetics of salbutamol sulfate, a commonly used β2-agonist in the treatment of asthma exacerbation, across Calu-3 respiratory epithelial cell monolayers in the presence of non-steroidal anti-inflammatory drugs (NSAIDs), as they have been implicated to be able to modulate organic cation transporters (OCTs). Methods. Calu-3 cell monolayers were grown in a liquid covered culture (LCC) configuration on 0.33 cm2 Transwell polyester cell culture supports. Monolayers, cultured between 11 and 14 days were evaluated for epithelial resistance, tight junction integrity, and expression of OCT using Western blot analysis. The transport of salbutamol across the monolayer was studied as a function of concentration. Directional transport was investigated by assessing apical–basal (a–b) and basal–apical (b-a) directions. The influence of a non-specific OCT inhibitor (tetraethylammonium, TEA) and three NSAIDs (aspirin, ibuprofen, and indomethacin) on the uptake of salbutamol was studied. Results. The flux of salbutamol sulfate increased with increasing concentration before reaching a plateau, suggesting the involvement of a transport-mediated uptake mechanism. Western blot analysis detected the presence of OCT1-3 and N1 and N2 sub-types, suggesting the presence of functioning transporters. The apparent permeability (Papp) of 0.1 mM salbutamol across the epithelial monolayer displayed directional transport in the a–b direction which was inhibited by ˜70% in the presence of TEA, suggesting OCT-mediated uptake. Likewise, the uptake of 0.1 mM salbutamol was decreased in the presence of all the three NSAIDs, supporting a mechanism whereby NSAIDs inhibit absorption of salbutamol across the bronchial epithelium via effects on the OCT transporters. Conclusion. This study demonstrates that NSAIDs influence the uptake kinetics of salbutamol in an in vitro Calu-3 cell system.

Fluticasone uptake across Calu-3 cells is mediated by salmeterol when deposited as a combination powder inhaler

We assessed whether co‐deposition of a long‐acting β2‐agonist and a corticosteroid affects their respective transport rates across epithelial cells.

The formulation of a pressurized metered dose inhaler containing theophylline for inhalation.

BACKGROUND Theophylline (TP) is a bronchodilator used orally to treat chronic obstructive pulmonary disease (COPD) that has been associated with multiple side effects, tempering its present use. This study aims to improve COPD treatment by creating a low-dose pressurized metered dose inhaler (pMDI) inhalable formulation of TP. METHODS Aerosol performance was assessed using Andersen Cascade Impaction (ACI). Solubility of TP in HFA 134/ethanol mixture was measured and morphology of the particles analyzed with a scanning electron microscope (SEM). Calu-3 cell viability, epithelial cell transport and inflammatory-response assays were conducted to study the impact of the formulation on lung epithelial cells. RESULTS The mass deposition profile of the formulation showed an emitted dose of 250.04±14.48μg per 5 actuations, achieving the designed nominal dose (50μg/dose). SEM showed that the emitted particles were hollow with spherical morphology. Approximately 98% of TP was transported across Calu-3 epithelial cells and the concentration of interleukin-8 secreted from Calu-3 cells following stimulation with tissue necrosis factor-α (TNF-α) resulted in significantly lower level of interleukin-8 released from the cells pre-treated with TP (1.92±0.77ng·ml(-1) TP treated vs. 8.83±2.05ng·ml(-1) TNF-α stimulated, respectively). CONCLUSIONS The solution pMDI formulation of TP developed in present study was shown to be suitable for inhalation and demonstrated anti-inflammatory effects at low doses in Calu-3 cell model.