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Human respiratory mucus is a biological hydrogel that forms a protective barrier for the underlying epithelium. Modulation of the mucus layer has been employed as a strategy to enhance transmucosal drug carrier transport. However, a drawback of this strategy is a potential reduction of the mucus barrier properties, in particular in situations with an increased exposure to particles. In this study, we investigated the impact of mucus modulation on its protective role. In vitro mucus was produced by Calu-3 cells, cultivated at the air-liquid interface for 21 days and used for further testing as formed on top of the cells. Analysis of confocal 3D imaging data revealed that after 21 days Calu-3 cells secrete a mucus layer with a thickness of 24 ± 6 μm. Mucus appeared to restrict penetration of 500 nm carboxyl-modified polystyrene particles to the upper 5–10 μm of the layer. Furthermore, a mucus modulation protocol using aerosolized N-acetylcysteine (NAC) was developed. This treatment enhanced the penetration of particles through the mucus down to deeper layers by means of the mucolytic action of NAC. These findings were supported by cytotoxicity data, indicating that intact mucus protects the underlying epithelium from particle-induced effects on membrane integrity. The impact of NAC treatment on the protective properties of mucus was probed by using 50 and 100 nm amine-modified and 50 nm carboxyl-modified polystyrene nanoparticles, respectively. Cytotoxicity was only induced by the amine-modified particles in combination with NAC treatment, implying a reduced protective function of modulated mucus. Overall, our data emphasize the importance of integrating an assessment of the protective function of mucus into the development of therapy approaches involving mucus modulation.

Remdesivir (RDV, GS-5734) is currently the only FDA-approved antiviral drug for the treatment of SARS CoV-2 infection. The drug is approved for use in adults or children 12-years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2 infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed and several including molnupiravir and PF-07321332 are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of Remdesivir nucleoside (RVn, GS-441524) that are processed to RVn-monophosphate, the precursor of the active RVn-triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells and Huh7.5 cells. In Syrian hamsters oral treatment with ODBG-P-RVn was well tolerated and achieved therapeutic levels in plasma above the EC90 for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.

The COVID-19 disease, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), emerged in late 2019 and rapidly spread worldwide, becoming a pandemic that infected millions of people and caused significant deaths. COVID-19 continues to be a major threat, and there is a need to deepen our understanding of the virus and its mechanisms of infection. To study the cellular responses to SARS-CoV-2 infection, we performed an RNA sequencing of infected vs. uninfected Calu-3 cells. Total RNA was extracted from infected (0.5 MOI) and control Calu-3 cells and converted to cDNA. Sequencing was performed, and the obtained reads were quality-analyzed and pre-processed. Differential expression was assessed with the EdgeR package, and functional enrichment was performed in EnrichR for Gene Ontology, KEGG pathways, and WikiPathways. A total of 1040 differentially expressed genes were found in infected vs. uninfected Calu-3 cells, of which 695 were up-regulated and 345 were down-regulated. Functional enrichment analyses revealed the predominant up-regulation of genes related to innate immune response, response to virus, inflammation, cell proliferation, and apoptosis. These transcriptional changes following SARS-CoV-2 infection may reflect a cellular response to the infection and help to elucidate COVID-19 pathogenesis, in addition to revealing potential biomarkers and drug targets. [ABSTRACT FROM AUTHOR]

The COVID-19 disease, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), emerged in late 2019 and rapidly spread worldwide, becoming a pandemic that infected millions of people and caused significant deaths. COVID-19 continues to be a major threat, and there is a need to deepen our understanding of the virus and its mechanisms of infection. To study the cellular responses to SARS-CoV-2 infection, we performed an RNA sequencing of infected vs. uninfected Calu-3 cells. Total RNA was extracted from infected (0.5 MOI) and control Calu-3 cells and converted to cDNA. Sequencing was performed, and the obtained reads were quality-analyzed and pre-processed. Differential expression was assessed with the EdgeR package, and functional enrichment was performed in EnrichR for Gene Ontology, KEGG pathways, and WikiPathways. A total of 1040 differentially expressed genes were found in infected vs. uninfected Calu-3 cells, of which 695 were up-regulated and 345 were down-regulated. Functional enrichment analyses revealed the predominant up-regulation of genes related to innate immune response, response to virus, inflammation, cell proliferation, and apoptosis. These transcriptional changes following SARS-CoV-2 infection may reflect a cellular response to the infection and help to elucidate COVID-19 pathogenesis, in addition to revealing potential biomarkers and drug targets.

Inflammation mediators enhance the activity of connexin (Cx) hemichannels, especially in the epithelial and endothelial tissues. As potential release routes for injury signals, such as (oligo)nucleotides, Cx hemichannels may contribute to long-lasting inflammation. Specific inhibition of Cx hemichannels may therefore be a mode of prevention and treatment of long-lasting, chronic sterile inflammation. The activity of Cx hemichannels was analysed in N2A and HeLa cells transfected with human Cx26 and Cx46 as well as in Calu-3 cells, using dye uptake as functional assay. Moreover, the possible impacts of the bioactive phenolic agents CVB2-61 and CVB4-57 on the barrier function of epithelial cells was analysed using Calu-3 cells. Both agents inhibited the dye uptake in N2A cells expressing Cx26 (>5 µM) and Cx46 (>20 µM). In Calu-3 cells, CVB2-61 and CVB4-57 reversibly inhibited the dye uptake at concentrations as low as 5 µM, without affecting the gap junction communication and barrier function, even at concentrations of 20 µM. While CVB2-61 or CVB4-57 maintained a reduced dye uptake in Calu-3 cells, an enhancement of the dye uptake in response to the stimulation of adenosine signalling was still observed after removal of the agents. The report shows that CVB2-61 and CVB4-57 reversibly block Cx hemichannels. Deciphering the mechanisms of the interactions of these agents with Cx hemichannels could allow further development of phenolic compounds to target Cx hemichannels for better and safer treatment of pathologies that involve Cx hemichannels. [ABSTRACT FROM AUTHOR]

Since human angiotensin-converting enzyme 2 (ACE2) serves as a primary receptor for SARS-CoV-2, characterizing ACE2 regions that allow SARS-CoV-2 to enter human cells is essential for designing peptide-based antiviral blockers and elucidating the pathogenesis of the virus. We identified and synthesized a 25-mer mimetic peptide (encompassing positions 22–46 of the ACE2 alpha-helix α1) implicated in the S1 receptor-binding domain (RBD)-ACE2 interface. The mimetic (wild-type, WT) ACE2 peptide significantly inhibited SARS-CoV-2 infection of human pulmonary Calu-3 cells in vitro. In silico protein modeling predicted that residues F28, K31, F32, F40, and Y41 of the ACE2 alpha-helix α1 are critical for the original, Delta, and Omicron strains of SARS-CoV-2 to establish the Spike RBD-ACE2 interface. Substituting these residues with alanine (A) or aspartic acid (D) abrogated the antiviral protective effect of the peptides, indicating that these positions are critical for viral entry into pulmonary cells. WT ACE2 peptide, but not the A or D mutated peptides, exhibited significant interaction with the SARS-CoV-2 S1 RBD, as shown through molecular dynamics simulations. Through identifying the critical amino acid residues of the ACE2 alpha-helix α1, which is necessary for the Spike RBD-ACE2 interface and mobilized during the in vitro viral infection of cells, we demonstrated that the WT ACE2 peptide protects susceptible K18-hACE2 mice against in vivo SARS-CoV-2 infection and is effective for the treatment of COVID-19. [Display omitted] • Protein modeling identified key amino acids in the ACE2 alpha-helix α1 involved in binding to the SARS-CoV-2 Spike. • A synthetic peptide from the ACE2 alpha-helix α1 protects Calu-3 human lung cells from SARS-CoV-2 infection in vitro. • This synthetic peptide also protects K18-hACE2 mice from SARS-CoV-2 infection. • The peptide helps treat lung inflammation caused by COVID-19 in K18-hACE2 mice. [ABSTRACT FROM AUTHOR]

Despite the fast development of vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still circulating and generating variants of concern (VoC) that escape the humoral immune response. In this context, the search for anti-SARS-CoV-2 compounds is still essential. A class of natural polyphenols known as flavonoids, frequently available in fruits and vegetables, is widely explored in the treatment of different diseases and used as a scaffold for the design of novel drugs. Therefore, herein we evaluate seven flavonoids divided into three subclasses, isoflavone (genistein), flavone (apigenin and luteolin) and flavonol (fisetin, kaempferol, myricetin, and quercetin), for COVID-19 treatment using cell-based assays and in silico calculations validated with experimental enzymatic data. The flavonols were better SARS-CoV-2 inhibitors than isoflavone and flavones. The increasing number of hydroxyl groups in ring B of the flavonols kaempferol, quercetin, and myricetin decreased the 50% effective concentration (EC50) value due to their impact on the orientation of the compounds inside the target. Myricetin and fisetin appear to be preferred candidates; they are both anti-inflammatory (decreasing TNF-α levels) and inhibit SARS-CoV-2 mainly by targeting the processability of the main protease (Mpro) in a non-competitive manner, with a potency comparable to the repurposed drug atazanavir. However, fisetin and myricetin might also be considered hits that are amenable to synthetic modification to improve their anti-SARS-CoV-2 profile by inhibiting not only Mpro, but also the 3′–5′ exonuclease (ExoN). [ABSTRACT FROM AUTHOR]

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S29. The “priming” of the surface S protein at S1/S2 (PR...RAR...685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S29 as KPSKR...815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-tocell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Cytokine storm is recognized as one of the factors contributing to organ failures and mortality in patients with COVID-19. Due to chronic inflammation, COVID-19 patients with diabetes mellitus (DM) or renal disease (RD) have more severe symptoms and higher mortality. However, the factors that contribute to severe outcomes of COVID-19 patients with DM and RD have received little attention. In an effort to investigate potential treatments for COVID-19, recent research has focused on the immunomodulation functions of mesenchymal stem cells (MSCs). In this study, the correlation between DM and RD and the severity of COVID-19 was examined by a combined approach with a meta-analysis and experimental research. The results of a systematic review and meta-analysis suggested that the odd of mortality in patients with both DM and RD was increased in comparison to those with a single comorbidity. In addition, in the experimental research, the data showed that high glucose and uremic toxins contributed to the induction of cytokine storm in human lung adenocarcinoma epithelial cells (Calu-3 cells) in response to SARS-CoV Peptide Pools. Of note, the incorporation of Wharton's jelly MSC-derived extracellular vesicles (WJ-EVs) into SARS-CoV peptide-induced Calu-3 resulted in a significant decrease in nuclear NF-κB p65 and the downregulation of the cytokine storm under high concentrations of glucose and uremic toxins. This clearly suggests the potential for WJ-EVs to reduce cytokine storm reactions in patients with both chronic inflammation diseases and viral infection. [ABSTRACT FROM AUTHOR]

Abstract Globally, many jurisdictions are legalizing or decriminalizing cannabis, creating a potential public health issue that would benefit from experimental evidence to inform policy, government regulations, and user practices. Tobacco smoke exposure science has created a body of knowledge that demonstrates the conclusive negative impacts on respiratory health; similar knowledge remains to be established for cannabis. To address this unmet need, we performed in vitro functional and transcriptomic experiments with a human airway epithelial cell line (Calu‐3) exposed to cannabis smoke, with tobacco smoke as a positive control. Demonstrating the validity of our in vitro model, tobacco smoke induced gene expression profiles that were significantly correlated with gene expression profiles from published tobacco exposure datasets from bronchial brushings and primary human airway epithelial cell cultures. Applying our model to cannabis smoke, we demonstrate that cannabis smoke induced functional and transcriptional responses that overlapped with tobacco smoke. Ontology and pathway analysis revealed that cannabis smoke induced DNA replication and oxidative stress responses. Functionally, cannabis smoke impaired epithelial cell barrier function, antiviral responses, and increased inflammatory mediator production. Our study reveals striking similarities between cannabis and tobacco smoke exposure on impairing barrier function, suppressing antiviral pathways, potentiating of pro‐inflammatory mediators, and inducing oncogenic and oxidative stress gene expression signatures. Collectively our data suggest that cannabis smoke exposure is not innocuous and may possess many of the deleterious properties of tobacco smoke, warranting additional studies to support public policy, government regulations, and user practices.

Background/Aims: Adenosine release and connexin (Cx) hemichannel activity are enhanced in the respiratory epithelium during pathophysiological events such as inflammation. We analysed the interplay between Cx channels and adenosine signalling in human respiratory airway epithelium using the Calu-3 cell line as a model. Methods: The Cx hemichannel activity in Calu-3 cells was evaluated by dye uptake assays. The expressed Cx isoforms and adenosine receptor subtypes were identified by PCR and western blot analysis. Pharmacological and molecular biological experiments were performed to analyse the involvement of the different adenosine receptor subtypes, the induced signalling pathways and the contribution of specific Cx isoforms to the hemichannel activity. Results: The adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased the dye uptake rate in Calu-3 cells. The pannexon and Cx hemichannel inhibitor carbenoxolone (CBX) did not supress the dye uptake at pannexin-specific concentrations (100 µM). High CBX concentrations or the inhibitor La3+, both effective on Cx hemichannels, were needed to inhibit the dye uptake. The NECA-related increase of dye uptake depended on enhanced cAMP synthesis and subsequent activation of the protein kinase A (PKA) as shown by quantification of cAMP levels and pharmacological inhibition of the adenylyl cyclase and the PKA. Further pharmacological inhibition as well as knockdown experiments with specific siRNA showed that the A2B adenosine receptor was the subtype mainly responsible for the increased dye uptake. The NECA-related increase of the dye uptake rate correlated with a decrease of Cx43 mRNA and an increase of Cx26 mRNA content in the cells as well as Cx26 protein synthesis and was inhibited by Cx26 knockdown using Cx26 siRNA. Of note, a siRNA-induced knockdown of Cx43 increased the content of Cx26 mRNA and correspondingly the dye uptake rate. Conclusion: The Calu-3 cell model shows that stimulation of the A2B adenosine receptor subtype activates synthesis of cAMP. cAMP activates PKA and induces thereby an increase in Cx26 and a decrease in Cx43 mRNA levels. As a result, the synthesis of Cx26 is reinforced, leading to an enhanced Cx hemichannel activity. The report identifies a mechanism that integrates adenosine release and Cx hemichannel activity and shows how adenosine signalling and Cx channels may act together to promote persistent inflammation, which is observed in several chronic diseases of the respiratory airway. [ABSTRACT FROM AUTHOR]

• The essential oil shows cytotoxicity as liquid and vapor phases. • The vapor phase exhibits strong cytotoxicity against Calu-3 cells. • Both necrosis and apoptosis are involved in Calu-3 cell death. • Lavandula dentata essential oil did not inhibit P-glycoprotein. • The vapor phase has potential as antitumor by inhalation route. The essential oil of Lavandula dentata L. possesses sedative, antioxidant, antibacterial, antifungal, carminative, antidepressant, and cytotoxic properties. The aims of this study were to investigate the cytotoxic effects of L. dentata essential oil on Calu-3 lung cancer cells in both vapor and liquid phases, and to provide further insights into its cytotoxic mechanisms. Usual MTT, SRB, and DNA-PI assays were used for evaluating cytotoxicity. In addition, LDH, acridine orange with ethidium bromide, Hoechst 33342, propidium iodide, Rhodamine 123, and Annexin V analyses were performed to explore the cytotoxic mechanisms. Rhodamine 123 accumulation assay was used for evaluating P-glycoprotein inhibitory potential. L. dentata essential oil provided a significant reduction of cell viability for all cytotoxic experiments by reaching 84% of cytotoxicity in vapor phase and showed a time-dependent profile. Both necrosis and apoptosis were involved in Calu-3 cell death. However, necrosis appeared to be the dominant cell death pathway. L. dentata essential oil did not inhibit P-glycoprotein. [ABSTRACT FROM AUTHOR]

Due to the continuing high impact of lung diseases on society and the emergence of new respiratory viruses, such as SARS-CoV-2, there is a great need for in vitro lung models that more accurately recapitulate the in vivo situation than current models based on lung epithelial cell cultures on stiff membranes. Therefore, we developed an in vitro airway epithelial–endothelial cell culture model based on Calu-3 human lung epithelial cells and human lung microvascular endothelial cells (LMVECs), cultured on opposite sides of flexible porous poly(trimethylene carbonate) (PTMC) membranes. Calu-3 cells, cultured for two weeks at an air–liquid interface (ALI), showed good expression of the tight junction (TJ) protein Zonula Occludens 1 (ZO-1). LMVECs cultured submerged for three weeks were CD31-positive, but the expression was diffuse and not localized at the cell membrane. Barrier functions of the Calu-3 cell cultures and the co-cultures with LMVECs were good, as determined by electrical resistance measurements and fluorescein isothiocyanate-dextran (FITC-dextran) permeability assays. Importantly, the Calu-3/LMVEC co-cultures showed better cell viability and barrier function than mono-cultures. Moreover, there was no evidence for epithelial- and endothelial-to-mesenchymal transition (EMT and EndoMT, respectively) based on staining for the mesenchymal markers vimentin and α-SMA, respectively. These results indicate the potential of this new airway epithelial–endothelial model for lung research. In addition, since the PTMC membrane is flexible, the model can be expanded by introducing cyclic stretch for enabling mechanical stimulation of the cells. Furthermore, the model can form the basis for biomimetic airway epithelial–endothelial and alveolar–endothelial models with primary lung epithelial cells.

Pulmonary delivery of sildenafil for the treatment of pulmonary arterial hypertension could overcome the limitations of intravenous and oral administration routes, such as poor patient compliance and systemic side effects. In this study, a carrier-free dry powder inhaler (DPI) formulation was developed, using spray drying technique and L-leucine as a dispersibility enhancer. Sildenafil citrate salt and sildenafil free base were evaluated for drug transport using a Calu-3 cell model, and their suitability for DPI production by spray drying was tested. Characteristics of the resultant carrier-free DPI powders were examined, namely crystallinity, morphology, size distribution, density, zeta potential, and aerodynamic performance. A Box-Behnken design was adopted to optimize the formulation and process conditions, including leucine amount, fraction of methanol in spraying solvent, and inlet temperature. While both sildenafil forms exhibited sufficient permeability for lung absorption, only sildenafil base resulted in DPI powders which were stable for 6 months. The introduction of leucine into the formulations effectively enhanced aerodynamic performance of the powders and particles with favorable size, shape, and density were produced. The optimal DPI formulation determined from experimental design possesses excellent aerodynamic performance with 89.39% emitted dose and 80.08% fine particle fraction, indicating the possibility of incorporating sildenafil into carrier-free DPIs for pulmonary delivery. [ABSTRACT FROM AUTHOR]

Highlights • A model system mimicking the lung lining was developed. • Surfactant protein D (SP-D) protects against C. albicans and A. fumigatus. • SP-D binds to these fungi and prevents fungal adhesion to the epithelium. • SP-D binding induces neutrophil accumulation and fungal clearance at the lining. Abstract At the lung lining innate defenses protect our lungs against inhaled fungal cells that could pose a threat to our health. These defenses are comprised of mucociliary clearance, soluble effector molecules and roaming phagocytic cells, such as macrophages and neutrophils. How important each of these defenses is during fungal clearance depends on the specific fungal pathogen in question and on the stage of infection. In this study the localization and antifungal activity of the lung surfactant protein D (SP-D) was studied in an environment mimicking the lung lining. To this end Calu-3 cells were grown on an air-liquid interface allowing them to polarize and to produce mucus at their apical surface. Additionally, neutrophils were added to study their role in fungal clearance. Two fungal pathogens were used for these experiments: Candida albicans and Aspergillus fumigatus , both of clinical relevance. During fungal infection SP-D localized strongly to both fungal surfaces and stayed bound through the different stages of infection. Furthermore, SP-D decreased fungal adhesion to the epithelium and increased fungal clearance by neutrophils from the epithelial surface. These findings suggest that SP-D plays an important role at the different stages of pulmonary defense against fungal intruders. [ABSTRACT FROM AUTHOR]

There is need to develop reproducible methods and experimental models for screening mucosal irritation and toxicity for drugs and pharmaceutical excipients. The aim of this study was to validate Calu-3 cell line as a model for screening respiratory irritation and toxicity of drugs and excipients. Eighteen test compounds were selected according to their irritation potential and European Centre for the Validation of Alternative Methods (ECVAM) guidelines. Cell toxicity and irritation was determined using MTT assay. Data analysis and interpretation were done using modified ECVAM approach; where replicate values met acceptance criteria if percent relative standard deviation (RSD) of the raw data is

The anti-inflammatory effect of polymeric deflazacort nanocapsules (NC-DFZ) was investigated, and possible improvement of epithelial barrier function using filter grown monolayers of Calu-3 cells was assessed. NC prepared from poly(ε-caprolactone) (PCL) had a mean size around 200 nm, slightly negative zeta potential (∼−8 mV), and low polydispersity index (<0.10). Encapsulation of DFZ had an efficiency of 85%. No cytotoxic effects were observed at particle concentration of 9.85 × 10 11 NC/ml, which was therefore chosen to evaluate the effect of NC-DFZ at 1% (w/v) of PCL and 0.5% (w/v) of DFZ on the epithelial barrier function of Calu-3 monolayers. Nanoencapsulated drug at 0.5% (w/v) increased transepithelial electrical resistance and decreased permeability of the paracellular marker sodium fluorescein, while non-encapsulated DFZ failed to improve these parameters. Moreover, NC-DFZ reduced the lipopolysaccharide (LPS) mediated secretion of the inflammatory marker IL-8. In vitro dissolution testing revealed controlled release of DFZ from nanocapsules, which may explain the improved effect of DFZ on the cells. These data suggest that nanoencapsulation of pulmonary delivered corticosteroids could be advantageous for the treatment of inflammatory conditions, such as asthma and chronic obstructive pulmonary diseases. [ABSTRACT FROM AUTHOR]

Pulmonary delivery of fluoroquinolones (FQs) is an interesting approach to treat lung infections as it may lead to high local concentrations while minimizing systemic exposure. However, FQs have a rapid diffusion through the lung epithelium giving the pulmonary route no advantage compared to the oral route. Interactions between FQs and metal cations form complexes which limit the diffusion through the epithelial barrier and would reduce the absorption of FQs and maintain high concentrations in the lung. The effects of this complexation depend on the FQ and the metal cations and optimum partners should be selected through in vitro experiments prior to aerosol drug formulation. In this study, CIP was chosen as a representative FQ and 5 cations (Ca 2 + , Mg 2 + , Zn 2 + , Al 3 + , Cu 2 + ) were selected to study the complexation and its effects on permeability, antimicrobial efficacy and cell toxicity. The results showed that the apparent association constants between CIP and cations ranked with the descending order: Cu 2 + > Al 3 + > Zn 2 + > Mg 2 + > Ca 2 + . When a target of 80% complexation was reached with the adequate concentrations of cations, the CIP permeability through the Calu-3 lung epithelial cells was decreased of 50%. Toxicity of the CIP on the Calu-3 cells, with an EC50 evaluated at 7 μM, was not significantly affected by the presence of the cations. The minimum inhibitory concentration of CIP for Pseudomonas aeruginosa was not affected or slightly increased in the range of cation concentrations tested, except for Mg 2 + . In conclusion, permeability was the main parameter that was affected by the metal cation complexation while cell toxicity and antimicrobial activity were not or slightly modified. Cu 2 + , with the highest apparent constant of association and with no effect on cell toxicity and antimicrobial activity of the CIP, appeared as a promising cation for the development of a controlled-permeability formulation of CIP for lung treatment. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the suitability of rhodamine-123, rhodamine-6G and rhodamine B as non-radioactive probes for characterizing organic cation transporters in respiratory cells. Fluorescent characteristics of the compounds were validated under standard in vitro drug transport conditions (buffers, pH, and light). Uptake/transport kinetics and intracellular accumulation of the compounds were investigated. Uptake/transport mechanisms were investigated by comparing the effect of pH, temperature, concentration, polarity, OCTs/OCTNs inhibitors/substrates, and metabolic inhibitors on the cationic dyes uptake in Calu-3 cells. Fluorescence stability and intensity of the compounds were altered by buffer composition, light, and pH. Uptake of the dyes was concentration-, temperature- and pH-dependent. OCTs/OCTNs inhibitors significantly reduced intracellular accumulation of the compounds. Whereas rhodamine-B uptake was sodium-dependent, pH had no effect on rhodamine-123 and rhodamine-6G uptake. Transport of the dyes across the cells was polarized: (AP → BL > BL → AP transport) and saturable: {V max = 14.08 ± 2.074, K m = 1821 ± 380.4 (rhodamine-B); V max = 6.555 ± 0.4106, K m = 1353 ± 130.4 (rhodamine-123) and V max = 0.3056 ± 0.01402, K m = 702.9 ± 60.97 (rhodamine-6G)}. The dyes were co-localized with MitoTracker®, the mitochondrial marker. Cationic rhodamines, especially rhodamine-B and rhodamine- 6G can be used as organic cation transporter substrates in respiratory cells. During such studies, buffer selection, pH and light exposure should be taken into consideration. [ABSTRACT FROM AUTHOR]

Purpose: An improved, simple apparatus was developed to easily and uniformly disperse dry powders onto an air-liquid interfaced cultured cell layer. We investigated drug permeation in cell cultures with access to the air-liquid interface (ALI) following deposition of a dry powder using the apparatus. Method: The improved apparatus for dispersing the powders was assembled. Dry powders containing model drugs were prepared and dispersed onto the cell layer with ALI. After the dispersion, the permeation of each model drug was measured and compared with other samples (solutions with the same compositions). Results: The improved apparatus could with ease uniformly disperse 40% of the loading dose onto the cell layer with ALI. Dry powders showed higher drug permeability compared to the samples. without cytotoxicity or an effect on tight junctions. The high drug permeability of dry powders was independent of the molecular weight of model drugs. The contribution of active transport was small, while an increase in passive drug transport via trans- and paracellular routes was observed. Conclusions: Inhaled dry powder formulations achieved higher drug permeability than their solution formulations in ALI. A high local concentration of drugs on the cell layer, caused by direct attachment of the inhaled dry powder, contributed to increased drug permeability via both trans- and paracellular routes. [ABSTRACT FROM AUTHOR]

Purpose: CriticalSorb™, with the principal component Solutol® HS15, is a novel mucosal drug delivery system demonstrated to improve the bioavailability of selected biotherapeutics. The intention of this study is to elucidate mechanism(s) responsible for the enhancement of trans-mucosal absorption of biological drugs by Solutol® HS15. Methods: Micelle size and CMC of Solutol® HS15 were determined in biologically relevant media. Polarised airway Calu-3 cell layers were used to measure the permeability of a panel of biological drugs, and to assess changes in TEER, tight junction and F-actin morphology. The rate of cell endocytosis was measured in vitro in the presence of Solutol® HS15 using a membrane probe, FM 2-10. Results: This work initially confirms surfactant-like behaviour of Solutol® HS15 in aqueous media, while subsequent experiments demonstrate that the effect of Solutol® HS15 on epithelial tight junctions is different from a 'classical' tight junction opening agent and illustrate the effect of Solutol® HS15 on the cell membrane (endocytosis rate) and F-actin cytoskeleton. Conclusion: Solutol® HS15 is the principle component of CriticalSorb™ that has shown an enhancement in permeability of medium sized biological drugs across epithelia. This study suggests that its mechanism of action arises primarily from effects on the cell membrane and consequent impacts on the cell cytoskeleton in terms of actin organisation and tight junction opening. [ABSTRACT FROM AUTHOR]

Background: Successful delivery of small interfering RNA (siRNA) to the lungs remains hampered by poor intracellular delivery, vector-mediated cytotoxicity, and an inability to withstand nebulization. Recently, a novel cyclodextrin (CD), SC12CDClickpropylamine, consisting of distinct lipophilic and cationic subunits, has been shown to transfect a number of cell types. However, the suitability of this vector for pulmonary siRNA delivery has not been assessed to date. To address this, a series of high-content analysis (HCA) and postnebulization assays were devised to determine the potential for CD-siRNA delivery to the lungs. Methods: SC12CDClickpropylamine-siRNA mass ratios (MRs) were examined for size and zeta potential. In-depth analysis of nanocomplex uptake and toxicity in Calu-3 bronchial epithelial cells was examined using IN Cell® HCA assays. Nebulized SC12CDClickpropylamine nanocomplexes were assessed for volumetric median diameter (VMD) and fine particle fraction (FPF) and compared with saline controls. Finally, postnebulization stability was determined by comparing luciferase knockdown elicited by SC12CDClickpropylamine nanocomplexes before and after nebulization. Results: SC12CDClickpropylamine-siRNA complexation formed cationic nanocomplexes of ≤200 nm in size depending on the medium and led to significantly higher levels of siRNA associated with Calu-3 cells compared with RNAiFect-siRNA-treated cells at all MRs ( p<0.001, n=3×4), with evidence of toxicity only at MRs 50-100. Nebulization of SC12CDClickpropylamine nanocomplexes using the Aeroneb® Pro resulted in VMDs of ∼4 μm and FPFs of ∼57% at all MRs. SC12CDClickpropylamine-siRNA-mediated luciferase knockdown was found to be 39.8±3.6% at MR=20 before and 35.6±4.55% after nebulization, comparable to results observed using unnebulized commercial transfection reagent, RNAiFect. Conclusions: SC12CDClickpropylamine nanocomplexes can be effectively nebulized for pulmonary delivery of siRNA using Aeroneb technology to mediate knockdown in airway cells. To the best of our knowledge, this is the first study examining the suitability of SC12CDClickpropylamine-siRNA nanocomplexes for pulmonary delivery. Furthermore, this work provides an integrated nanomedicine-device combination for future in vitro and in vivo preclinical and clinical studies of inhaled siRNA therapeutics. [ABSTRACT FROM AUTHOR]

There is need to develop reproducible methods and experimental models for screening mucosal irritation and toxicity for drugs and pharmaceutical excipients. The aim of this study was to validate Calu-3 cell line as a model for screening respiratory irritation and toxicity of drugs and excipients. Eighteen test compounds were selected according to their irritation potential and European Centre for the Validation of Alternative Methods (ECVAM) guidelines. Cell toxicity and irritation was determined using MTT assay. Data analysis and interpretation were done using modified ECVAM approach; where replicate values met acceptance criteria if percent relative standard deviation (RSD) of the raw data is <18%. Compounds with mean relative viability values of 50% and below were classified as irritant (I); those above 50% were non-irritant (NI). At low concentration (0.2% w/v) and 1 h incubation, the Calu-3 cell culture model accurately predicted the toxicity of most test compounds. The specificity of our proposed model (percentage of in vivo non-irritants correctly predicted), concordance (percentage of compounds correctly predicted) and sensitivity (percentage of in vivo irritants correctly predicted) at 0.2% w/v and 60 min exposure were 100%, 72%, and 44%, respectively. In conclusion, the Calu-3 cell line in conjunction with MTT assay appears to be a potentially useful tool for screening drugs and excipients for respiratory mucosa irritation and toxicity. However, as the data reported in this study were solely based on MTT assay, additional studies are needed using other toxicity-/irritation-indicating methods to confirm the observed trend. [ABSTRACT FROM AUTHOR]

We describe a compartmentalized microdevice specifically designed to perform permeability studies across a model of lung barrier. Epithelial cell barriers were reproduced by culturing Calu-3 cells at the air-liquid interface (AIC) in 1 mm microwells made from a perforated glass slide with an embedded porous membrane. We created a single basolateral reservoir for all microwells which eliminated the need to renew the growth medium during the culture growth phase. To perform drug permeability studies on confluent cell layers, the cell culture slide was aligned and joined to a collection platform consisting in 35 μL collection reservoirs connected at the top and bottom with microchannels. The integrity and functionality of the cell barriers were demonstrated by measurement of trans-epithelial electrical resistance (TEER), confocal imaging and permeability assays of C-sucrose. Micro-cell barriers were able to form confluent layers in 1 week, demonstrating a similar bioelectrical evolution as the Transwell systems used as controls. Tight junctions were observed throughout the cell-cell interfaces, and the low permeability coefficients of C-sucrose confirmed their functional presence, creating a primary barrier to the diffusion of solutes. This microdevice could facilitate the monitoring of biomolecule transport and the screening of formulations promoting their passage across the pulmonary barrier, in order to select candidates for pulmonary administration to patients. [ABSTRACT FROM AUTHOR]

Broad area of respiratory epithelium with mild surface conditions is an attractive possibility when trans-mucosal delivery of protein drugs is considered. A mucus and cellular barrier of respiratory epithelium can be modelled in vitro by Calu-3 cell line. We have monitored morphology and barrier properties of Calu-3 culture on permeable supports while developing into liquid covered or air interfaced and mucus lined cellular barrier. Besides morphological differences, cultures differed in electrical resistance and permeability to proteins as well. The accelerated permeability to proteins in these models, due to permeability modulator MP C16, was examined. The effect on electrical resistance of cellular layer was rapid in both cultures suggesting easy access of MP C16 to cells even though its overall impact on cell permeability was strongly reduced in mucus covered culture. Differences in properties of the two models enable better understanding of protein transmucosal permeability, suggesting route of transport and MP C16 modulator action. [ABSTRACT FROM AUTHOR]

Abstract: Non-invasive delivery of biotherapeutics, as an attractive alternative to injections, could potentially be achieved through the mucosal surfaces, utilizing nanoscale therapeutic carriers. However, nanoparticles do not readily cross the mucosal barriers, with the epithelium presenting a major barrier to their translocation. The transcytotic pathway of vitamin B12 has previously been shown to ‘ferry’ B12-decorated nanoparticles across intestinal epithelial (Caco-2) cells. However, such studies have not been reported for the airway epithelium. Furthermore, the presence in the airways of the cell machinery responsible for transepithelial trafficking of B12 is not widely reported. Using a combination of molecular biology and immunostaining techniques, our work demonstrates that the bronchial cell line, Calu-3, expresses the B12-intrinsic factor receptor, the transcobalamin II receptor and the transcobalamin II carrier protein. Importantly, the work showed that sub-200nm model nanoparticles chemically conjugated to B12 were internalised and transported across the Calu-3 cell layers, with B12 conjugation not only enhancing cell uptake and transepithelial transport, but also influencing intracellular trafficking. Our work therefore demonstrates that the B12 endocytotic apparatus is not only present in this airway model, but also transports ligand-conjugated nanoparticles across polarised epithelial cells, indicating potential for B12-mediated delivery of nanoscale carriers of biotherapeutics across the airways. [Copyright &y& Elsevier]

Abstract: In a number of pulmonary diseases, patients may develop abnormally viscous mucus reducing drug efficacy. To increase budesonide diffusion within lung fluid, we developed nanoporous microparticles (NPMPs) composed of budesonide and a mucokinetic, ambroxol hydrochloride, to be inhaled as a dry powder. Budesonide/ambroxol-HCl particles were formulated by spray drying and characterised by various physicochemicals methods. Aerodynamic properties were evaluated using a cascade impactor. Drugs apparent permeability coefficients were calculated across mucus producing Calu-3 cell monolayers cultivated at an air–liquid interface. Microparticles made only from budesonide and ambroxol-HCl had smooth surfaces. In the presence of ammonium carbonate ((NH4)2CO3), NPMPs were formulated, with significantly (P <0.05) superior aerodynamic properties (MMAD=1.87±0.22μm and FPF=84.0±2.6%). The formation of nanopores and the increase in the specific surface area in the presence of (NH4)2CO3 were mainly attributed to the neutralisation of ambroxol-HCl to form ambroxol base. Thus, ambroxol base could behave in the same manner as budesonide and prompt nanoprecipitation when spray dried from an ethanol/water mix occurs. All formulations were amorphous, which should enhance dissolution rate and diffusion through lung fluid. These NPMPs were able to improve budesonide permeability across mucus producing Calu-3 cell monolayers (P <0.05) suggesting that they should be able to enhance budesonide diffusion in the lungs through viscous mucus. [Copyright &y& Elsevier]

Purpose: We sought to identify key variables in cellular architecture and physiology that might explain observed differences in the passive transport properties of small molecule drugs across different airway epithelial cell types. Methods: Propranolol (PR) was selected as a weakly basic, model compound to compare the transport properties of primary (NHBE) vs. tumor-derived (Calu-3) cells. Differentiated on Transwell™ inserts, the architecture of pure vs. mixed cell co-cultures was studied with confocal microscopy followed by quantitative morphometric analysis. Cellular pharmacokinetic modeling was used to identify parameters that differentially affect PR uptake and transport across these two cell types. Results: Pure Calu-3 and NHBE cells possessed different structural and functional properties. Nevertheless, mixed Calu-3 and NHBE cell co-cultures differentiated as stable cell monolayers. After measuring the total mass of PR, the fractional areas covered by Calu-3 and NHBE cells allowed deconvoluting the transport properties of each cell type. Based on the apparent thickness of the unstirred, cell surface aqueous layer, local differences in the extracellular microenvironment explained the measured variations in passive PR uptake and permeation between Calu-3 and NHBE cells. Conclusion: Mixed cell co-cultures can be used to compare the local effects of the extracellular microenvironment on drug uptake and transport across two epithelial cell types. [ABSTRACT FROM AUTHOR]

Alkylglycoside surfactants have been proposed as drug delivery excipients with the potential to enhance mucosal drug absorption of therapeutic macromolecules. Previous work reported their drug absorption-promoting potential by demonstrating that several compounds within this class of surfactants improve mucosal absorption of peptides, proteins and other macromolecules. However, detailed investigation of their toxicity has not been conducted. Using Calu-3 epithelial cell layers as a model of the airway mucosa, and liposomes as models of cell membranes, this work investigates the cytotoxicity of dodecylmaltoside, tridecylmaltoside and tetradecylmaltoside, as representative alkylglycosides. A combination of different toxicity assays and other tests indicating cell membrane disruption were used to assess cytotoxicity. The alkylglycosides tested induced a dramatic reduction in cell viability, cell membrane and liposome-disruptive effects, as well as abrogation of transepithelial electrical resistance that did not recover completely. Importantly, these phenomena were noted at concentrations markedly lower than those typically used in the literature studies demonstrating the absorption-enhancing properties of alkylglycosides. This work therefore demonstrates that alkylglycosides exhibit significant toxicity towards airway epithelial cells, most likely resulting from a membrane-damaging effect, highlighting a need for further evaluation of their safety as absorption-enhancing excipients. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:114-125, 2013 [ABSTRACT FROM AUTHOR]

Abstract: A physiologically pertinent in vitro model is urgently needed for probing interactions between inhaled drugs and the organic cation transporters (OCT) in the bronchial epithelium. This study evaluated OCT expression, functionality, inhibition by common inhaled drugs and impact on formoterol transepithelial transport in layers of human bronchial epithelial Calu-3 cells grown at an air–liquid interface. 21 day old Calu-3 layers expressed OCT1, OCT3, OCTN1 and OCTN2 whereas OCT2 could not be detected. Quantification of the cellular uptake of the OCT substrate ASP+ in presence of inhibitors suggested several OCT were functional at the apical side of the cell layers. ASP+ uptake was reduced by the bronchodilators formoterol, salbutamol (albuterol), ipratropium and the glucocorticoid budesonide. However, the OCT inhibitory properties of the two β2-mimetics were suppressed at therapeutically relevant concentrations. The absorptive permeability of formoterol across the cell layers was enhanced at a high drug concentration shown to decrease ASP+ uptake by ∼50% as well as in presence of the OCT inhibitor tetraethylammonium (TEA). Secretory transport was unaffected by the drug concentration but was reduced by TEA. Our data indicate air-interfaced Calu-3 layers offer a low-cost in vitro model suitable for assessing inhaled drug–OCT interactions in the bronchial epithelium. [Copyright &y& Elsevier]

Abstract: Wheat germ agglutinin (WGA) modified PEG–PLA nanoparticles (WGA–NP) have demonstrated its potential for enhancing delivery of peptides into brain following intranasal administration. However, the effect of ligand density is less well known. WGA density may affect nanoparticles uptake in nasal epithelial cells through ligand-receptor interactions, and the damage to nasal tissue since WGA showed cytotoxicity to cells in a dose-dependent manner. In this study, the effect of WGA density on WGA–NP was studied with regard to both the uptake and toxicity in vitro, using Calu-3 cells, which express a number of N-acetylglucosamine on their cell surface. Nanoparticles containing different WGA ligand densities were prepared by controlling the molar ratio of thiolated WGA to maleimide–PEG–PLA (WGA/maleimide) and particles properties were examined. With the increase of WGA/maleimide ratio, the particle size, WGA density and the hemoagglutination increased, while the conjugation efficiency decreased. The in vitro study showed markedly enhanced endocytosis of WGA–NP compared to NP in Calu-3 cells and significant inhibition of uptake in the presence of chitin. Cytotoxicity of WGA–NP increased gradually with the increase of molar ratio of WGA to maleimide, nanoparticles concentration and incubation time. WGA–NP showed the highest efficiency of uptake and a mild cytotoxicity when the molar ratio of WGA to maleimide was 1:10. These results suggest that WGA density plays an important role in both cellular uptake and toxicity of WGA–NP via a receptor-mediated mechanism. Therefore, to achieve a more rational approach of drug delivery system design, the surface density of the targeting moiety on the nanoparticles surface should be considered. [Copyright &y& Elsevier]

Abstract: The aim of the present study was to investigate whether Calu-3 cell culture conditions influence drug and nutrient transport known to occur via carriers or transporters. Calu-3 cell layers, an in vitro model of the lung epithelium, were cultured using air interfaced culture (AIC) or liquid covered culture (LCC) on either polycarbonate or polyester as filter support material. We found that the development of the Calu-3 cell layer barrier function did not depend on the filter material but rather on the culture conditions as follows: (i) the apical uptake of Gly-Sar was significantly larger for cells grown in AIC compared to LCC, (ii) the TEER values for cells grown in LCC were approximately three times larger than for cells grown in AIC, (iii) the transepithelial transport in both AIC and LCC Calu-3 cells was polarized in the apical–basolateral direction of proline, glycine, α-methyl-d-glucoside, glipizide, taurocholic acid and estrone-3-sulfate, whereas inulin, mannitol and Gly-Sar showed no polarized transport. Etoposide showed polarized efflux (basolateral to apical transport) in AIC and LCC Calu-3 layers. These findings provide information about nutrient and drug transport in Calu-3 cells, and this may have implications for selecting culture conditions for transport studies in this in vitro model of the lung epithelium. [Copyright &y& Elsevier]

Abstract: Absorption studies with aerosol formulation delivered by metered dose inhalers across cell- and tissue-based in vitro models of the pulmonary epithelia are not trivial due to the complexity of the processes involved: (i) aerosol generation and deposition, (ii) drug release from the carrier, and (iii) absorption across the epithelial air–blood barrier. In contrast to the intestinal mucosa, pulmonary epithelia are only covered by a thin film of lining fluid. Submersed cell culture systems would not allow to studying the deposition of aerosol particles and their effects on this delicate epithelial tissue. We developed a new Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) to mimic the inhalation of a single metered aerosol dose and its subsequent deposition on filter-grown pulmonary epithelial cell monolayers exposed to an air–liquid interface. The reproducibility of deposition of these dry powder aerosols and subsequent drug transport across Calu-3 monolayers with commercially available dry powder inhalers containing salbutamol sulphate or budesonide could be demonstrated. In the context of developing new dry powder aerosol formulations, PADDOCC appears as a useful tool, allowing reducing animal testing and faster translation into clinical trials. [ABSTRACT FROM AUTHOR]

Abstract: Amphotericin B (AMB), a potent antifungal agent, has been employed as an inhalable therapy for pulmonary fungal infections. We recently described a novel nano-sized delivery vehicle composed of phospholipid (PL) and apolipoprotein A-I, NanoDisk (ND), to which we added AMB as a payload (ND-AMB). The goal of the present study was to evaluate whether ND-AMB, compared to other formulations, preserves lung cell integrity in vitro, as AMB can be toxic to mammalian cells and reduce lung function when inhaled. Epithelial integrity was assessed by measuring K+ ion flux across a model airway epithelium, Calu-3 cells. In this assay ND-AMB was at least 8-fold less disruptive than AMB/deoxycholate (DOC). Cell viability studies confirmed this observation. Unexpectedly, the ND vehicle restored the integrity of a membrane compromised by prior exposure to AMB. An alternative formulation of ND-AMB containing a high load of AMB per ND was not protective, suggesting that ND with a low ratio of AMB to PL can sequester additional AMB from membranes. ND-AMB also protected HepG2 cells from the cytotoxicity of AMB, as determined by cellular viability and lactate dehydrogenase (LDH) levels. This study suggests that ND-AMB may be safe for administration via inhalation and reveals a unique activity whereby ND-AMB protects lung epithelial membranes from AMB toxicity. [Copyright &y& Elsevier]

The aim of this study was to use different toxicity indices to investigate the effect of N, N-Dimethylacetamide (DMA), Polyethylene glycol 400 (PEG 400), Methyl-Pyrrolidone/aromatic hydrocarbon (MPH), Cremophor® EL, and Dimethylsulfoxide (DMSO) on Calu-3 cells. Membrane perturbation and cytotoxicity were investigated using lactate dehydrogenase (LDH), 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolinium bromide (MTT), transepithelial electrical resistance (TEER) assessment, sodium fluorescein (SF) permeation, and phalloidin actin staining. The MDH activity of cells treated with DMSO (≤ 4.0 v/v), Cremophor EL (≤ 10% v/v), and PEG 400 (≤ 10% v/v) was not significantly altered ( p > 0.5). Similarly, DMSO (≤ 8.0% v/v), Cremophor® EL (≤ 16.0% v/v) and PEG 400 (≤ 32.0% v/v) had no significant effect on LDH ( p > 0.05). Conversely, MPH and DMA at very low concentrations (≤ 0.25% v/v) induced cell toxicity. However, up to 2.0% v/v MPH and (DMA) had no effect on TEER and SF permeation. No obvious change in actin staining was observed for DMSO (15%), Cremophor® EL (15%), and MPA (1%). However, the actin architecture for cells treated with DMA (1%) and PEG 400 (15%) appeared significantly different from control. Based on this study Cremophor (≤ 10%), PEG 400 (≤ 5%), and DMSO (≤ 5%), and low concentrations of DMA and MPA (≤ 0.25%) were suitable for in vitro studies with Calu-3 cells. [ABSTRACT FROM AUTHOR]

The role of transporters in drug absorption, distribution and elimination processes as well as in drug–drug interactions is increasingly being recognised. Although the lungs express high levels of both efflux and uptake drug transporters, little is known of the implications for the biopharmaceutics of inhaled drugs. The current knowledge of the expression, localisation and functionality of drug transporters in the pulmonary tissue and the few studies that have looked at their impact on pulmonary drug absorption is extensively reviewed. The emphasis is on transporters most likely to affect the disposition of inhaled drugs: (1) the ATP-binding cassette (ABC) superfamily which includes the efflux pumps P-glycoprotein (P-gp), multidrug resistance associated proteins (MRPs), breast cancer resistance protein (BCRP) and (2) the solute-linked carrier (SLC and SLCO) superfamily to which belong the organic cation transporter (OCT) family, the peptide transporter (PEPT) family, the organic anion transporter (OAT) family and the organic anion transporting polypeptide (OATP) family. Whenever available, expression and localisation in the intact human tissue are compared with those in animal lungs and respiratory epithelial cell models in vitro. The influence of lung diseases or exogenous agents on transporter expression is also mentioned. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2240–2255, 2010 [ABSTRACT FROM AUTHOR]

Abstract: Cyclodextrins (CDs) are promising solubility enhancers for inhaled drug delivery. However, they have dose-dependent effects on the respiratory epithelium, which may have advantages for permeability enhancement but also gives rise to safety concerns. In this study, the methyl thiazol tetrazolium (MTT) assay was used to compare a new sparingly methylated β-CD, Kleptose® Crysmeβ (Crysmeb) with the more established CD derivatives hydroxypropyl-γ-cyclodextrin (HPγCD), randomly methylated β-cyclodextrin (Rameb) and hydroxypropyl-β-cyclodextrin (HPβCD). The βCD derivatives affected cell metabolism in A549 cells in a concentration dependent manner with LD50 of 56, 31 and 11 mM obtained for HPβCD, Crysmeb and Rameb, respectively. Calu-3 cells were less susceptible to βCD with an LD50 of 25 mM being obtained for Rameb only. Permeability increases in Calu-3 cell layers were observed with βCD derivatives and a concentration dependency shown. The mechanism of permeability enhancement and its reversibility was investigated. Rameb produced an irreversible loss of cell layer barrier function at ≥25 mM, but perturbations of epithelial integrity were moderate and reversible in the case of HPβCD and Crysmeb (25–50 mM). Given its high solubilisation capacity, the low toxicity and transient absorption promoting properties, this study identifies Crysmeb as a promising adjuvant in formulations for inhalation. [Copyright &y& Elsevier]

Abstract: We report the effects of new N-acylated ambroxol derivatives (TEI-588a, TEI-588b, TEI-589a, TEI-589b, TEI-602a and TEI-602b: a, aromatic amine-acylated derivative; b, aliphatic amine-acylated derivative) induced from ambroxol (a mucolytic agent to treat human lung diseases) on Cl− secretion in human submucosal serous Calu-3 cells under a Na+/K+/2Cl− cotransporter-1 (NKCC1)-mediated hyper-secreting condition. TEI-589a, TEI-589b and TEI-602a diminished hyper-secretion of Cl− by diminishing the activity of NKCC1 without blockade of apical Cl− channel (TEI-589a>TEI-602a>TEI-589b), while any other tested compounds including ambroxol had no effects on Cl− secretion. These indicate that the inhibitory action of an aromatic amine-acylated derivative on Cl− secretion is stronger that that of an aliphatic amine-acylated derivative, and that 3-(2,5-dimethyl)furoyl group has a strong action in inhibition of Cl− secretion than cyclopropanoyl group. We here indicate that TEI-589a, TEI-589b and TEI-602a reduce hyper-secretion to an appropriate level in the airway, providing a possibility that the compound can be an effective drug in airway obstructive diseases including COPD by reducing the airway resistance under a hyper-secreting condition. [Copyright &y& Elsevier]

Abstract: The main goal of the present study was to investigate the potential of a new generation of hybrid polysaccharide nanocarriers, composed of chitosan (CS) and anionic cyclodextrins (CDs), for gene delivery to the airway epithelium. More specifically, these nanocarriers were investigated with regard to their ability to enter epithelial cells and promote gene expression in the Calu-3 cell culture model. In the search for the most suitable nanocarrier composition for gene delivery, the effect of CS molecular weight (Mw) on the nanocarriers characteristics and their ability to transfect cells was investigated. Thus, hybrid CS/CD nanoparticles were prepared with two different CS Mw, medium (110kDa) and low (10kDa), and loaded with pSEAP (plasmid DNA model that encodes the expression of secreted alkaline phosphatase). The resulting nanoparticles presented an adequate size range (100–200nm, depending on CS Mw), a positive surface charge (+22 to +35mV) and very high DNA association efficiency values (>90%). Cellular uptake studies showed that the nanoparticles were effectively internalized by the cells, providing a good indication of their potential as gene carriers. The transfection efficiency of the different formulations, measured by the concentration of secreted gene product (SEAP), indicated that all the nanoparticles were able to elicit a significantly higher response than the naked DNA (control), the transfection efficiency being more important for low MwCS nanoparticles than for those composed of medium MwCS. Overall, this report is the first evidence of the potential of a new generation of safe polysaccharide nanocarriers for gene delivery to the airway epithelium. [Copyright &y& Elsevier]