Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro.

Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.

An Enteric-Coated Polyelectrolyte Nanocomplex Delivers Insulin in Rat Intestinal Instillations when Combined with a Permeation Enhancer.

The use of nanocarriers is being researched to achieve oral peptide delivery. Insulin-associated anionic polyelectrolyte nanoparticle complexes (PECs) were formed that comprised hyaluronic acid and chitosan in an optimum mass mixing ratio of 5:1 (MR 5), followed by coating with a pH-dependent polymer. Free insulin was separated from PECs by size exclusion chromatography and then measured by HPLC. The association efficiency of insulin in PECs was >95% and the loading was ~83 µg/mg particles. Dynamic light scattering and nanoparticle tracking analysis of PECs revealed low polydispersity, a negative zeta potential range of -40 to -50 mV, and a diameter range of 95-200 nm. Dissolution studies in simulated small intestinal fluid (FaSSIF-V2) revealed that the PECs were colloidally stable. PECs that were coated with Eudragit® L-100 delayed insulin release in FaSSIF-V2 and protected insulin against pancreatin attack more than uncoated PECs. Uncoated anionic PECs interacted weakly with mucin in vitro and were non-cytotoxic to Caco-2 cells. The coated and uncoated PECs, both concentrated further by ultrafiltration, permitted dosing of 50 IU/kg in rat jejunal instillations, but they failed to reduce plasma glucose or deliver insulin to the blood. When ad-mixed with the permeation enhancer (PE), sucrose laurate (100 mM), the physicochemical parameters of coated PECs were relatively unchanged, however blood glucose was reduced by 70%. In conclusion, the use of a PE allowed for the PEC-released bioactive insulin to permeate the jejunum. This has implications for the design of orally delivered particles that can release the payload when formulated with enhancers.

Saponin Micelles Lead to High Mucosal Permeation and In Vivo Efficacy of Solubilized Budesonide.

Due to fast nasal mucociliary clearance, only the dissolved drug content can effectively permeate the mucosa and be pharmaceutically active after intranasal application of suspensions. Therefore, the aim of this study was to increase the budesonide concentration in solution of a nasal spray formulation. Budesonide, a highly water-insoluble corticosteroid, was successfully solubilized using a micellar formulation comprising escin, propylene glycol and dexpanthenol in an aqueous buffered environment ("Budesolv"). A formulation based on this micellar system was well-tolerated in the nasal cavity as shown in a good laboratory practice (GLP) local tolerance study in rabbits. Ex vivo permeation studies into porcine nasal mucosa revealed a faster and more efficient absorption. Budesolv with 300 µg/mL solubilized budesonide resulted in a budesonide concentration of 42 µg/g tissue after only 15 min incubation. In comparison, incubation with the marketed product Rhinocort® aqua 64 (1.28 mg/mL budesonide as suspension) led to 15 µg/g tissue. The in vivo tumor-necrosis-factor (TNF)-α secretion in an acute lung inflammation mouse model was significantly reduced (p < 0.001) following a prophylactic treatment with Budesolv compared to Rhinocort® aqua 64. Successful treatment 15 min after the challenge was only possible with Budesolv (40% reduction of TNF-α, p = 0.0012) suggesting a faster onset of action. The data reveal that solubilization based on saponin micelles presents an opportunity for the development of products containing hardly soluble substances that result in a faster onset and a better topical treatment effect.

Nanoparticle Tracking Analysis to Examine the Temperature-Induced Aggregation of Proteins.

In recent years, nanoparticle tracking analysis (NTA) has emerged as an alternative tool for particle size characterization. Especially when examining polydisperse systems, individual particle to particle tracking allows for higher peak resolution than dynamic light scattering techniques. However, NTA requires an experienced user with a good insight into how the different settings can affect the determination of particle size and size distributions. This chapter provides a guideline for protein aggregation studies using the example of temperature-induced aggregation of IgG at low concentration.

Intra-articular delivery of a nanocomplex comprising salmon calcitonin, hyaluronic acid, and chitosan using an equine model of joint inflammation.

Polyelectrolyte nanoparticle constructs (NPs) comprising salmon calcitonin (sCT), chitosan (CS), and hyaluronic acid (HA) were previously established as having anti-inflammatory potential when injected via the intra-articular (i.a.) route to a mouse model. We attempted to translate the formulation to a large animal model, the lipopolysaccharide (LPS)-stimulated equine model of joint inflammation. The aim was to manufacture under aseptic conditions to produce sterile pyrogen-free NPs, to confirm physicochemical characteristics, and to test toxicity and efficacy in a pilot study. NP dispersions were successfully formulated using pharmaceutical-grade source materials and were aseptically manufactured under GMP-simulated conditions in a grade A modular aseptic processing workstation. The NP formulation had no detectable pathogen or endotoxin contamination. NPs were then tested versus a lactated Ringer's solution control following single i.a. injections to the radiocarpal joints of two groups of four horses pre-treated with LPS, followed by arthrocentesis at set intervals over 1 week. There was no evidence of treatment-related toxicity over the period. While there were no differences between clinical read-outs of the NP and the control, two synovial fluid-derived biomarkers associated with cartilage turnover revealed a beneficial effect of NPs. In conclusion, NPs comprising well-known materials were manufactured for an equine i.a.-injectable pilot study and yielded no NP-attributable toxicity. Evidence of NP-associated benefit at the level of secondary endpoints was detected as a result of decreases in synovial fluid inflammatory biomarkers.