µCT to quantify muco-obstructive lung disease and effects of neutrophil elastase knockout in mice.

Muco-obstructive lung diseases are characterized by airway obstruction and hyperinflation, which can be quantified by imaging. Our aim was to evaluate µCT for longitudinal quantification of muco-obstructive lung disease in ß-epithelial Na+ channel overexpressing (Scnn1b-TG) mice and of the effects of neutrophil elastase (NE) knockout on its progression. Lungs from wild-type (WT), NE-/-, Scnn1b-TG, and Scnn1b-TG/NE-/- mice were scanned with 9-µm resolution at 0, 5, 14, and 60 days of age, and airway and parenchymal disease was quantified. Mucus adhesion lesions (MAL) were persistently increased in Scnn1b-TG compared with WT mice from 0 days (20.25 ± 6.50 vs. 9.60 ± 2.07, P < 0.05), and this effect was attenuated in Scnn1b-TG/NE-/- mice (5.33 ± 3.67, P < 0.001). Airway wall area percentage (WA%) was increased in Scnn1b-TG mice compared with WT from 14 days onward (59.2 ± 6.3% vs. 49.8 ± 9.0%, P < 0.001) but was similar in Scnn1b-TG/NE-/- compared with WT at 60 days (46.4 ± 9.2% vs. 45.4 ± 11.5%, P = 0.97). Air proportion (Air%) and mean linear intercept (Lm) were persistently increased in Scnn1b-TG compared with WT from 5 days on (53.9 ± 4.5% vs. 30.0 ± 5.5% and 78.82 ± 8.44 µm vs. 65.66 ± 4.15 µm, respectively, P < 0.001), whereas in Scnn1b-TG/NE-/-, Air% and Lm were similar to WT from birth (27.7 ± 5.5% vs. 27.2 ± 5.9%, P = 0.92 and 61.48 ± 9.20 µm vs. 61.70 ± 6.73 µm, P = 0.93, respectively). Our results suggest that µCT is sensitive to detect the onset and progression of muco-obstructive lung disease and effects of genetic deletion of NE on morphology of airways and lung parenchyma in Scnn1b-TG mice, and that it may serve as a sensitive endpoint for preclinical studies of novel therapeutic interventions for muco-obstructive lung diseases.

Increased Inflammatory Markers Detected in Nasal Lavage Correlate with Paranasal Sinus Abnormalities at MRI in Adolescent Patients with Cystic Fibrosis.

Chronic rhinosinusitis (CRS) is a characteristic feature of cystic fibrosis (CF) multiorgan disease and develops early in the life of patients with CF. The study aimed to correlate the inflammatory markers and the presence of structural abnormalities detected by MRI in the paranasal sinuses of patients with CF. Methods: Nasal lavage and MRI of the paranasal sinuses was performed in a cohort of 30 CF patients (median age 14 y; range 7-20 y). Morphological abnormalities characteristic of CF were evaluated with a dedicated CRS MRI scoring system and correlated with different inflammation parameters measured in nasal lavage. Inflammation of the paranasal sinuses was positively associated with structural abnormalities in MRI. The concentration of the pro-inflammatory markers neutrophil elastase (NE) and the neutrophil elastase/alpha1-antitrypsin (NE/A1AT) complex correlated significantly with CRS-MRI sum score (p < 0.05, r = 0.416 and p < 0.05, r = 0.366, respectively). S. aureus infection was associated with the increased pro-inflammatory cytokine activity of IL-6 and IL-8, and increased levels of NE/A1AT complex in our patients (p < 0.05, respectively). CRS-MRI sum score and individual sinus MRI scores were positively associated with inflammatory activity as a sign of CRS pathology present in CF.

Chronic rhinosinusitis with nasal polyps is associated with impaired TMEM16A-mediated epithelial chloride secretion.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is one of the most common chronic disorders with limited therapeutic options. However, the pathogenesis of CRSwNP remains poorly understood. OBJECTIVE: We sought to determine the role of abnormalities in nasal epithelial ion transport in primary epithelial cultures and patients with CRSwNP. METHODS: We studied epithelial ion transport and transcript levels of the Cl- channels cystic fibrosis transmembrane conductance regulator and transmembrane protein 16A (TMEM16A) in human primary nasal epithelial cultures of patients with CRSwNP and healthy controls. Furthermore, we determined expression levels of proinflammatory cytokines that have been implicated in the regulation of epithelial ion channels (IL-1ß, INF-γ, TNF-α, IL-13) and studied effects of the key TH2 signaling molecule IL-13 in CRSwNP and control nasal epithelial cultures. Finally, we measured in vivo nasal potential difference to compare epithelial ion transport in patients with CRSwNP and controls. RESULTS: Bioelectric studies demonstrated that Ca2+-activated Cl- secretion was reduced in CRSwNP versus control nasal epithelial cultures. Transcript levels of IL-13 and the Ca2+-activated Cl- channel TMEM16A were increased in CRSwNP cultures. Stimulation with IL-13 increased TMEM16A expression further and restored Ca2+-activated Cl- secretion in CRSwNP cultures. Nasal potential difference measurements demonstrated reduced Ca2+-activated Cl- transport in patients with CRSwNP versus controls. CONCLUSIONS: This study demonstrates that TMEM16A-mediated Ca2+-activated Cl- secretion is reduced in primary nasal epithelial cultures and nasal epithelia of patients with CRSwNP. Our data suggest that the Ca2+-activated Cl- channel TMEM16A may be implicated in the pathogenesis and serve as a novel therapeutic target in patients with CRSwNP.

Organic Cation Transporters in the Lung-Current and Emerging (Patho)Physiological and Pharmacological Concepts.

Organic cation transporters (OCT) 1, 2 and 3 and novel organic cation transporters (OCTN) 1 and 2 of the solute carrier 22 (SLC22) family are involved in the cellular transport of endogenous compounds such as neurotransmitters, l-carnitine and ergothioneine. OCT/Ns have also been implicated in the transport of xenobiotics across various biological barriers, for example biguanides and histamine receptor antagonists. In addition, several drugs used in the treatment of respiratory disorders are cations at physiological pH and potential substrates of OCT/Ns. OCT/Ns may also be associated with the development of chronic lung diseases such as allergic asthma and chronic obstructive pulmonary disease (COPD) and, thus, are possible new drug targets. As part of the Special Issue "Physiology, Biochemistry and Pharmacology of Transporters for Organic Cations", this review provides an overview of recent findings on the (patho)physiological and pharmacological functions of organic cation transporters in the lung.

Physicochemical and biopharmaceutical characterization of novel Matrix-Liposomes.

Matrix-Liposomes (MLs) are a very promising solid oral drug delivery system; however, data on their interaction with biological membranes are not available. Here, we describe the quality of MLs manufactured by dual centrifugation. MLs were prepared with a Z-average range of 139 to 160 nm and a PDI of 0.18 to 0.25. To investigate the effect of MLs on intestinal tissue (with and without mucolytic treatment), we then established an ex vivo rat intestine model. The integrity of the epithelial membranes of rat intestine was not affected by the incubation with MLs without or with pre-mucolytic treatment. Tissue samples were also analysed for changes in P-glycoprotein (P-gp) expression and function. The net secretion of the P-gp substrate Rh123 across the rat duodenum was increased in the presence of MLs. To summarize, MLs do not affect intestinal epithelial integrity, although they impact Rh123 secretion. In future, these novel MLs have to be further evaluated for proficient intestinal drug delivery.

OCTN2-Mediated Acetyl-l-Carnitine Transport in Human Pulmonary Epithelial Cells In Vitro.

The carnitine transporter OCTN2 is associated with asthma and other inflammatory diseases. The aims of this work were (i) to determine carnitine uptake into freshly isolated human alveolar type I (ATI)-like epithelial cells in primary culture, (ii) to compare the kinetics of carnitine uptake between respiratory epithelial in vitro cell models, and (iii) to establish whether any cell line was a suitable model for studies of carnitine transport at the air-blood barrier. Levels of time-dependent [3H]-acetyl-l-carnitine uptake were similar in ATI-like, NCl-H441, and Calu-3 epithelial cells, whereas uptake into A549 cells was ~5 times higher. Uptake inhibition was more pronounced by OCTN2 modulators, such as l-Carnitine and verapamil, in ATI-like primary epithelial cells compared to NCl-H441 and Calu-3 epithelial cells. Our findings suggest that OCTN2 is involved in the cellular uptake of acetyl-l-carnitine at the alveolar epithelium and that none of the tested cell lines are optimal surrogates for primary cells.

Identification of the Tetraspanin CD9 as an Interaction Partner of Organic Cation Transporters 1 and 2.

Organic cation transporters (OCTs) are membrane proteins with relevant physiological (because they accept neurotransmitters as substrate) and pharmacological (because of their interaction with drugs) roles. The human OCTs hOCT1 (SLC22A1/hOCT1) and hOCT2 (SLC22A2/hOCT2) are highly expressed in hepatic (hOCT1) and in renal and neuronal tissue (hOCT2), suggesting a possible role in modulating neurotransmitter activity in the liver, kidney, and brain, and their clearance from the blood. Even though there are several data demonstrating that OCTs are regulated under various patho-physiological conditions, it remains largely unknown which proteins directly interact with OCTs and thereby influence their cellular processing, localization, and function. In this work, using a mating-based split-ubiquitin yeast two-hybrid system, we characterized the potential interactome of hOCT1 and 2. It became evident that these OCTs share some potential interaction partners, such as the tetraspanins CD63 and CD9. Moreover, we confirmed interaction of hOCT2 with CD9 by fluorescence-activated cell sorting coupled with Förster resonance energy transfer analysis. Together with other proteins, tetraspanins build "tetraspanins webs" in the plasma membrane, which are able to regulate cellular trafficking and compartmentalization of interacting partners. While CD63 was demonstrated to mediate the localization of the hOCT2 to the endosomal system, we show here that co-expression of hOCT2 and CD9 led to strong cell surface localization of the transporter. These data suggest that tetraspanins regulate the cellular localization and function of OCTs. Co-localization of CD9 and hOCT was confirmed in tissues endogenously expressing proteins, highlighting the potential biological relevance of this interaction.

Expression and function of Anoctamin 1/TMEM16A calcium-activated chloride channels in airways of in vivo mouse models for cystic fibrosis research.

Physiological processes of vital importance are often safeguarded by compensatory systems that substitute for primary processes in case these are damaged by gene mutation. Ca2+-dependent Cl- secretion in airway epithelial cells may provide such a compensatory mechanism for impaired Cl- secretion via cystic fibrosis transmembrane conductance regulator (CFTR) channels in cystic fibrosis (CF). Anoctamin 1 (ANO1) Ca2+-gated Cl- channels are known to contribute to calcium-dependent Cl- secretion in tracheal and bronchial epithelia. In the present study, two mouse models of CF were examined to assess a potential protective function of Ca2+-dependent Cl- secretion, a CFTR deletion model (cftr-/-), and a CF pathology model that overexpresses the epithelial Na+ channel ß-subunit (ßENaC), which is encoded by the Scnn1b gene, specifically in airway epithelia (Scnn1b-Tg). The expression levels of ANO1 were examined by mRNA and protein content, and the channel protein distribution between ciliated and non-ciliated epithelial cells was analyzed. Moreover, Ussing chamber experiments were conducted to compare Ca2+-dependent Cl- secretion between wild-type animals and the two mouse models. Our results demonstrate that CFTR and ANO1 channels were co-expressed with ENaC in non-ciliated cells of mouse tracheal and bronchial epithelia. Ciliated cells did not express these proteins. Despite co-localization of CFTR and ANO1 in the same cell type, cells in cftr-/- mice displayed no altered expression of ANO1. Similarly, ANO1 expression was unaffected by ßENaC overexpression in the Scnn1b-Tg line. These results suggest that the CF-related environment in the two mouse models did not induce ANO1 overexpression as a compensatory system.

Bypassing CFTR dysfunction in cystic fibrosis with alternative pathways for anion transport.

One therapeutic strategy for cystic fibrosis (CF) seeks to restore anion transport to affected epithelia by targeting other apical membrane Cl- channels to bypass dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. The properties and regulation of the Ca2+-activated Cl- channel TMEM16A argue that long-acting small molecules which target directly TMEM16A are required to overcome CFTR loss. Through genetic studies of lung diseases, SLC26A9, a member of the solute carrier 26 family of anion transporters, has emerged as a promising target to bypass CFTR dysfunction. An alternative strategy to circumvent CFTR dysfunction is to deliver to CF epithelia artificial anion transporters that shuttle Cl- across the apical membrane. Recently, powerful, non-toxic, biologically-active artificial anion transporters have emerged.

Cellular distribution and function of ion channels involved in transport processes in rat tracheal epithelium.

Transport of water and electrolytes in airway epithelia involves chloride-selective ion channels, which are controlled either by cytosolic Ca2+ or by cAMP The contributions of the two pathways to chloride transport differ among vertebrate species. Because rats are becoming more important as animal model for cystic fibrosis, we have examined how Ca2+- dependent and cAMP- dependent Cl- secretion is organized in the rat tracheal epithelium. We examined the expression of the Ca2+-gated Cl- channel anoctamin 1 (ANO1), the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, the epithelial Na+ channel ENaC, and the water channel aquaporin 5 (AQP5) in rat tracheal epithelium. The contribution of ANO1 channels to nucleotide-stimulated Cl- secretion was determined using the channel blocker Ani9 in short-circuit current recordings obtained from primary cultures of rat tracheal epithelial cells in Ussing chambers. We found that ANO1, CFTR and AQP5 proteins were expressed in nonciliated cells of the tracheal epithelium, whereas ENaC was expressed in ciliated cells. Among nonciliated cells, ANO1 occurred together with CFTR and Muc5b and, in addition, in a different cell type without CFTR and Muc5b. Bioelectrical studies with the ANO1-blocker Ani9 indicated that ANO1 mediated the secretory response to the nucleotide uridine-5'-triphosphate. Our data demonstrate that, in rat tracheal epithelium, Cl- secretion and Na+ absorption are routed through different cell types, and that ANO1 channels form the molecular basis of Ca2+-dependent Cl- secretion in this tissue. These characteristic features of Cl--dependent secretion reveal similarities and distinct differences to secretory processes in human airways.

Generation and functional characterization of epithelial cells with stable expression of SLC26A9 Cl- channels.

Recent studies identified the SLC26A9 Cl(-) channel as a modifier and potential therapeutic target in cystic fibrosis (CF). However, understanding of the regulation of SLC26A9 in epithelia remains limited and cellular models with stable expression for biochemical and functional studies are missing. We, therefore, generated Fisher rat thyroid (FRT) epithelial cells with stable expression of HA-tagged SLC26A9 via retroviral transfection and characterized SLC26A9 expression and function using Western blotting, immunolocalization, whole cell patch-clamp, and transepithelial bioelectric studies in Ussing chambers. We demonstrate stable expression of SLC26A9 in transfected FRT (SLC26A9-FRT) cells on the mRNA and protein level. Immunolocalization and Western blotting detected SLC26A9 in different intracellular compartments and to a lesser extent at the cell surface. Whole cell patch-clamp recordings demonstrated significantly increased constitutive Cl(-) currents in SLC26A9-FRT compared with control-transduced FRT (Control-FRT) cells (P < 0.01). Similar, transepithelial measurements showed that the basal short circuit current was significantly increased in SLC26A9-FRT vs. Control-FRT cell monolayers (P < 0.01). SLC26A9-mediated Cl(-) currents were increased by cAMP-dependent stimulation (IBMX and forskolin) and inhibited by GlyH-101, niflumic acid, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), as well as RNAi knockdown of WNK1 implicated in epithelial osmoregulation. Our results support that these novel epithelial cells with stable expression of SLC26A9 will be a useful model for studies of pharmacological regulation including the identification of activators of SLC26A9 Cl(-) channels that may compensate deficient cystic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) secretion and serve as an alternative therapeutic target in patients with CF and potentially other muco-obstructive lung diseases.

Organic cation transporter function in different in vitro models of human lung epithelium.

Organic cation transporters (OCT) encoded by members of the solute carrier (SLC) 22 family of genes are involved in the disposition of physiological substrates and xenobiotics, including drugs used in the treatment of chronic obstructive lung diseases and asthma. The aim of this work was to identify continuously growing epithelial cell lines that closely mimic the organic cation transport of freshly isolated human alveolar type I-like epithelial cells (ATI) in primary culture, and which consequently, can be utilised as in vitro models for the study of organic cation transport at the air-blood barrier. OCT activity was investigated by measuring [(14)C]-tetraethylammonium (TEA) uptake into monolayers of Calu-3, NCI-H441 and A549 lung epithelial cell lines in comparison to ATI-like cell monolayers in primary culture. Levels of time-dependent TEA uptake were highest in A549 and ATI-like cells. In A549 cells, TEA uptake had a saturable and a non-saturable component with Km=528.5±373.1µM, Vmax=0.3±0.1nmol/min/mg protein and Kd=0.02µl/min/mg protein. TEA uptake into Calu-3 and NCI-H441 cells did not reach saturation within the concentration range studied. RNAi experiments in A549 cells confirmed that TEA uptake was mainly facilitated by OCT1 and OCT2. Co-incubation studies using pharmacological OCT modulators suggested that organic cation uptake pathways share several similarities between ATI-like primary cells and the NCI-H441 cell line, whereas more pronounced differences exist between primary cells and the A549 and Calu-3 cell lines.

Beta-2 Adrenergic Agonists Are Substrates and Inhibitors of Human Organic Cation Transporter 1.

Beta-2-adrenergic agonists are first line therapeutics in the treatment of asthma and chronic obstructive pulmonary disease (COPD). Upon inhalation, bronchodilation is achieved after binding to ß2-receptors, which are primarily localized on airway smooth muscle cells. Given that ß2-adrenergic agonists chemically are bases, they carry net positive charge at physiologic pH value in the lungs (i.e., pH 7.4). Here, we studied whether ß2-agonists interact with organic cation transporters (OCT) and whether this interaction exerted an influence on their passage across the respiratory epithelium to their target receptors. [14C]-TEA uptake into proximal (i.e., Calu-3) and distal (i.e., A549 and NCI-H441) lung epithelial cells was significantly reduced in the presence of salbutamol sulfate, formoterol fumarate, and salmeterol xinafoate in vitro. Expression of all five members of the OCT/N family has been confirmed in human pulmonary epithelial cells in situ and in vitro, which makes the identification of the transporter(s) responsible for the ß2-agonist interaction challenging. Thus, additional experiments were carried out in HEK-293 cells transfected with hOCT1-3. The most pronounced inhibition of organic cation uptake by ß2-agonists was observed in hOCT1 overexpressing HEK-293 cells. hOCT3 transfected HEK-293 cells were affected to a lesser extent, and in hOCT2 transfectants only marginal inhibition of organic cation uptake by ß2-agonists was observed. Bidirectional transport studies across confluent NCI-H441 cell monolayers revealed a net absorptive transport of [3H]-salbutamol, which was sensitive to inhibition by the OCT1 modulator, verapamil. Accordingly, salbutamol uptake into hOCT1 overexpressing HEK-293 cells was time- and concentration-dependent and could be completely blocked by decynium-22. Taken together, our data suggest that ß2-agonists are specific substrates and inhibitors of OCT1 in human respiratory epithelial cells and that this transporter might play a role in the pulmonary disposition of drugs of this class.

The cell line NCl-H441 is a useful in vitro model for transport studies of human distal lung epithelial barrier.

The lack of a well characterized, continuously growing in vitro model of human distal lung epithelial phenotype constitutes a serious limitation in the area of inhalation biopharmaceutics, particularly in the context of transepithelial transport studies. Here, we investigated if a human lung adenocarcinoma cell line, NCl-H441, has potential to serve as an in vitro model of human distal lung epithelium. The development of barrier properties was studied by immunocytochemistry (ICC) against the junction proteins zonula occludens protein 1 (ZO-1) and E-cadherin and measurement of transepithelial electrical resistance (TEER). Moreover, transport studies with the paracellular marker compounds fluorescein sodium and fluorescein isothiocyanate (FITC)-labeled dextrans of molecular weights ranging from 4 to 70 kDa were carried out. The expression of P-glycoprotein (P-gp; ABCB1) and organic cation transporters (OCT/Ns; SLC22A1-A5) was investigated by ICC and immunoblot. P-gp function was assessed by monolayer release and bidirectional transport studies using rhodamine 123 (Rh123) and the inhibitors verapamil and LY335979. Uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) was measured, in order to assess organic cation transporter function in vitro. Furthermore, the inhibitory potential of several organic cations on ASP(+) uptake was studied. NCl-H441 cells, when grown under liquid-covered conditions, formed confluent, electrically tight monolayers with peak TEER values of approximately 1000 Ω·cm(2), after 8-12 days in culture. These monolayers were able to differentiate paracellularly transported substrates according to their molecular weight. Presence of P-gp, OCT1, OCT2, OCT3, OCTN1, and OCTN2 was confirmed by Western blot and ICC and was similar to data from freshly isolated human alveolar epithelial cells in primary culture. Rh123 release from NCI-H441 monolayers was time-dependent and showed low, albeit significant attenuation by both inhibitors. In transport studies, Rh123 exhibited net secretion, which again was inhibitable by bona fide P-gp modulators. The uptake of ASP(+) was time- and temperature-dependent with Km = 881.2 ± 195.3 µM and Vmax = 2.07 ± 0.26 nmol/min/mg protein. TEA, amantadine, quinidine, and verapamil significantly inhibited ASP(+) uptake into NCl-H441 cells, whereas the effect of d- and l-carnitine and ergothioneine, two OCTN substrates, was less pronounced. NCl-H441 cells are the first cell line of human distal lung epithelial origin with the ability to form monolayers with appreciable barrier properties. Moreover, drug transporter expression and activity in NCl-H441 cells was consistent with what has been reported for human alveolar epithelial cells in primary culture.

The verapamil transporter expressed in human alveolar epithelial cells (A549) does not interact with ß2-receptor agonists.

  Affinity of different organs for verapamil is highly variable and organ-specific. For example, the drug exhibits high levels of accumulation in lung tissues. A transporter recognising verapamil as a substrate has previously been identified in human retinal pigment epithelial (RPE) and in rat retinal capillary endothelial (TR-iBRB2) cells. This transporter is distinct from any of the cloned organic cation transporters. Therefore, we hypothesised that the verapamil transporter is also functionally expressed in the human respiratory mucosa. Moreover, we tested the hypothesis that this transporter interacts with pulmonary administered cationic drugs such as ß2-agonists. The uptake of [(3)H]verapamil was studied in A549 human alveolar epithelial cell monolayers at different times and concentrations. The influence of extracellular proton concentration and various organic cations on verapamil uptake was determined. Verapamil uptake into A549 cells was time- and concentration-dependent, sensitive to pH and had a Km value of 39.8 ± 8.2 µM. Verapamil uptake was also sensitive to inhibition by amantadine, quinidine and pyrilamine, but insensitive to other typical modulators of organic cation and choline transporters. Whilst we demonstrated functional activity of the elusive verapamil transporter at the lung epithelium, our data suggest that this transporter does not interact with ß2-agonists at therapeutic concentrations.

Biopharmaceutical in vitro characterization of CPZEN-45, a drug candidate for inhalation therapy of tuberculosis.

BACKGROUND: The caprazamycin derivative, CPZEN-45 has previously demonstrated antitubercular activity against Mycobacterium tuberculosis H37Rv. Here, the authors report a basic biopharmaceutical characterization of the compound focusing on in vitro permeability and cytotoxicity, with respect to the suitability of CPZEN-45 hydrochloride for inhalation treatment of tuberculosis. RESULTS: MTT assays confirmed that CPZEN-45 HCl had no acute cytotoxic effects up to 3 mg/ml. In transport studies, apparent permeability coefficients of CPZEN-45 HCl across Calu-3 monolayers in absorptive and secretive directions were 0.43 ± 0.20 × 10(-6) cm/s and 0.38 ± 0.12 × 10(-6) cm/s, respectively. Across ATI-like monolayers, apparent permeability values were 12.10 ± 4.31 × 10(-6) cm/s and 8.50 ± 1.83 × 10(-6) cm/s. CPZEN-45 HCl formed colloidal complexes at concentrations above 0.38 mg/ml; however, these complexes were not micelles, as assessed by Orange OT encapsulation assay. CONCLUSION: CPZEN-45 is an interesting new drug candidate with potential to be used in aerosol therapy of tuberculosis.

Organic cation transporters in the blood-air barrier: expression and implications for pulmonary drug delivery.

Studies concerning the impact that hepatic, renal and intestinal transporters have on drug disposition have been frequently reported in the literature. Surprisingly, however, little is known regarding the distribution and function of drug-transporter proteins of the lung epithelium. Many drugs (delivered to the lung) have a net positive charge and, thus, are potential substrates of organic cation transporters; currently marketed compounds (e.g., bronchodilators), as well as novel drug candidates in development, are such substrates. It is the aim of this review to summarize the current state of organic cation-transporter expression analysis in the lung and in in vitro models of bronchial and alveolar barriers. Moreover, activity of selected transporters in lung epithelium in situ and in vitro will be highlighted, and their potential role in pulmonary drug disposition will be addressed. One example included here is the transporter-dependent absorption of beta2-agonists in respiratory epithelial cells.

Transport of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) in human respiratory epithelial cells.

Organic cation/carnitine transporters (OCT/N) mediate uptake of positively charged molecules. Their role in lung epithelium; however, is not well understood. OCT/N expression and activity was studied in cell lines of human alveolar (A549), bronchial (16HBE14o- and Calu-3) and intestinal (Caco-2) epithelium. Protein levels were largely comparable for all OCT/Ns in the respiratory epithelial cell lines studied; however, OCT2 was exclusively observed in A549 cells. OCT1 and -2 were present at significantly higher levels in Caco-2 cells, compared with the pulmonary epithelial cell types. OCTN1 and -2 were also more abundant in Caco-2. Only OCT3 was expressed evenly across all cell lines investigated. Uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) was dependent on concentration, temperature, membrane potential and pH. In 16HBE14o-, Calu-3 and Caco-2 monolayers, substrate saturation of ASP(+) uptake was not reached. Alveolar A549 cells showed saturable ASP(+) uptake via two transporter sites with K(m) values of 12.5 ± 4.0 µM and 456.9 ± 164.5 µM, respectively. This uptake was sensitive to organic cations, but insensitive to carnitine and lysine. We conclude that uptake of organic cations is facilitated by distinct pathways in different regions of lung mucosa. Luminally localised OCT2 appears to be exclusively involved in the alveolar epithelium, whereas basolateral localised OCT3 might play a role in alveolar as well as in bronchial epithelial cells.

In vitro and in vivo characterisation of PEG-lipid-based micellar complexes of salmon calcitonin for pulmonary delivery.

PURPOSE: To investigate DSPE-PEG(2000)-based micellar formulations of salmon calcitonin (sCT) for their ability to improve pulmonary delivery. METHODS: Micelles were characterised by DLS and (31)P-NMR spectroscopy. Stability against sCT degrading peptidases, trypsin, α-chymotrypsin and neutrophil elastase as well as their influence on transepithelial absorption was investigated in vitro. In vivo performance of sCT micelles was studied in an experimental model of intratracheal aerosolisation into rats. RESULTS: Micelles with a mean hydrodynamic diameter of 12 nm spontaneously assembled, when a total concentration of 0.02 mM of PEG-lipid and sCT (at 1:1 molar ratio) was exceeded. Nuclear magnetic resonance confirmed the presence of small micellar structures. The micellar formulation showed increased stability against enzymatic digestion. In vitro studies also showed that sCT micelles were able to enhance transepithelial absorption. Data obtained from in vivo experiments provided evidence of significantly (P < 0.05) higher mean plasma concentrations of sCT, after inhalation of micelles compared to sCT solution, at 60 and 90 min, a significantly higher AUC (inf) and a relative bioavailability of 160 ± 55% when compared to plain sCT solution. CONCLUSIONS: The herein described PEG-lipid micelles are promising carriers for enhanced pulmonary delivery of sCT.

New fluorescent bile acids: synthesis, chemical characterization, and disastereoselective uptake by Caco-2 cells of 3-deoxy 3-NBD-amino deoxycholic and ursodeoxycholic acid.

Deoxycholic acid (DCA), a secondary bile acid (BA), and ursodeoxycholic acid (UDCA), a tertiary BA, cause opposing effects in vivo and in cell suspensions. Fluorescent analogues of DCA and UDCA could help investigate important questions about their cellular interactions and distribution. We have prepared a set of isomeric 3α- and 3ß-amino analogues of UDCA and DCA and derivatised these with the discrete fluorophore, 4-nitrobenzo-2-oxa-1,3-diazol (NBD), forming the corresponding four fluorescent adducts. These absorb in the range 465-470 nm and fluoresce at approx. 535 nm. In order to determine the ability of the new fluorescent bile acids to mimic the parents, their uptake was studied using monolayers of Caco-2 cells, which are known to express multiple proteins of the organic anion-transporting peptide (OATP) subfamily of transporters. Cellular uptake was monitored over time at 4 and 37°C to distinguish between passive and active transport. All four BA analogues were taken up but in a strikingly stereo- and structure-specific manner, suggesting highly discriminatory interactions with transporter protein(s). The α-analogues of DCA and to a lesser extent UDCA were actively transported, whereas the ß-analogues were not. The active transport process was saturable, with Michaelis-Menten constants for 3α-NBD DCA (5) being K(m)=42.27±12.98 µM and V(max)=2.8 ± 0.4 nmol/(mg protein*min) and for 3α-NBD UDCA (3) K(m)=28.20 ± 7.45 µM and V(max)=1.8 ± 0.2 nmol/(mg protein*min). These fluorescent bile acids are promising agents for investigating questions of bile acid biology and for detection of bile acids and related organic anion transport processes.