Evaluation of in vitro rat and human airway epithelial models for acute inhalation toxicity testing.

In vivo models (mostly rodents) are currently accepted by regulatory authorities for assessing acute inhalation toxicity. Considerable efforts have been made in recent years to evaluate in vitro human airway epithelial models (HAEM) as replacements for in vivo testing. In the current work, an organotypic in vitro rat airway epithelial model (RAEM), rat EpiAirway, was developed and characterized to allow a direct comparison with the available HAEM, human EpiAirway, in order to address potential interspecies variability in responses to harmful agents. The rat and human models were evaluated in 2 independent laboratories with 14 reference chemicals, selected to cover a broad range of chemical structures and reactive groups, as well as known acute animal and human toxicity responses, in 3 replicate rounds of experiments. Toxicity endpoints included changes in tissue viability (MTT assay), epithelial barrier integrity (TEER, transepithelial electrical resistance), and tissue morphology (histopathology). The newly developed rat EpiAirway model produced reproducible results across all replicate experiments in both testing laboratories. Furthermore, a high level of concordance was observed between the RAEM and HAEM toxicity responses (determined by IC25) in both laboratories, with R2=0.78 and 0.88 when analyzed by TEER; and R2=0.92 for both when analyzed by MTT. These results indicate that rat and human airway epithelial tissues respond similarly to acute exposures to chemicals. The new in vitro RAEM will help extrapolate to in vivo rat toxicity responses and support screening as part of a 3Rs program.

Increasing Cellular Uptake and Permeation of Curcumin Using a Novel Polymer-Surfactant Formulation.

Several therapeutically active molecules are poorly water-soluble, thereby creating a challenge for pharmaceutical scientists to develop an active solution for their oral drug delivery. This study aimed to investigate the potential for novel polymer-surfactant-based formulations (designated A and B) to improve the solubility and permeability of curcumin. A solubility study and characterization studies (FTIR, DSC and XRD) were conducted for the various formulations. The cytotoxicity of formulations and commercial comparators was tested via MTT and LDH assays, and their permeability by in vitro drug transport and cellular drug uptake was established using the Caco-2 cell model. The apparent permeability coefficients (Papp) are considered a good indicator of drug permeation. However, it can be argued that the magnitude of Papp, when used to reflect the permeability of the cells to the drug, can be influenced by the initial drug concentration (C0) in the donor chamber. Therefore, Papp (suspension) and Papp (solution) were calculated based on the different values of C0. It was clear that Papp (solution) can more accurately reflect drug permeation than Papp (suspension). Formulation A, containing Soluplus® and vitamin E TPGs, significantly increased the permeation and cellular uptake of curcumin compared to other samples, which is believed to be related to the increased aqueous solubility of the drug in this formulation.

Evaluation of the toxicity of sodium dodecyl sulphate (SDS) in the MucilAir™ human airway model in vitro.

The aim of the study was to use multiple in vitro assays to assess the effects of a model irritant, sodium dodecyl sulphate (SDS) (≤10 mM (0.29 %, w/v)), on an in vitro model of the airway, MucilAir™. The use of MucilAir™ in recovery studies was also explored. A 24 h exposure increased IL-8 release at an SDS concentration ≥0.63 mM (0.018 %, w/v). Mucin secretion increased and transepithelial electrical resistance (TEER) decreased at SDS concentrations ≥1.25 mM (0.04 %, w/v). Cytotoxicity (lactate dehydrogenase (LDH) release into basolateral chamber) was observed at SDS concentrations of ≥2.5 mM (0.07 %, w/v). The sensitivity of the assays was IL-8 release > TEER = mucin secretion > LDH release. After 7 days, full or partial recovery was observed for intermediate concentrations of SDS using all assays but not at 5 and 10 mM SDS. Morphologically, erosion and cell loss were observed at these concentrations. Resazurin metabolism at 7 days tended to decrease in a dose-dependent manner at SDS concentrations above 2.5 mM (0.07 %, w/v). Together, these data support a No Observable Effect Level of 0.31 mM (0.009 % w/v) SDS and the use of MucilAir™ as a relevant model for airway toxicity studies.

The effect of ingredients commonly used in nasal and inhaled solutions on the secretion of mucus in vitro.

Hypersecretion of mucus is associated with impaired mucociliary clearance that can influence the retention of active pharmaceutical ingredients in the airway but is also linked with recurrent airway disease. Therefore, the effect on mucin secretion of a range of ingredients used in solutions delivered to the nose and lung was studied. Mucin secretion from explants of ovine epithelium was quantified using an enzyme-linked lectin assay (ELLA) or sandwich ELLA depending on the compatibility of the ingredients with the assay. Benzalkonium chloride (0.015% w/w), Methocel™ E50 premium LV (1.0% w/w), propylene glycol (1.5% w/w), potassium sorbate + propylene glycol (0.3% w/w + 1.5% w/w) and polysorbate 80 (0.025% w/w), used at common working concentrations, all increased the secretion of mucin from the explants (P < 0.05). Ethylenediamine tetraacetic acid-disodium salt (EDTA) (0.015% w/w), Avicel® RC591 (1.5% w/w), fluticasone furoate (0.0004% w/w, concentration in solution) and dimethyl sulfoxide (DMSO) (0.2% w/w) did not affect mucin secretion. Compounds increasing mucin secretion could alter the rate of mucociliary clearance and the mucus could provide a barrier to drug absorption. This could predispose patients to disease and affect the activity of delivered drugs, decreasing or increasing their clinical efficacy.

A comparison of three mucus-secreting airway cell lines (Calu-3, SPOC1 and UNCN3T) for use as biopharmaceutical models of the nose and lung.

The aim of this work was to compare three existing mucus-secreting airway cell lines for use as models of the airways to study drug transport in the presence of mucus. Each cell line secreted mature, glycosylated mucins, evidenced by the enzyme-linked lectin assay. The secretagogue, adenylyl-imidodiphosphate, increased mucin secretion in SPOC1 (3.5-fold) and UNCN3T (1.5-fold) cells but not in Calu-3 cells. In a novel mucus-depleted (MD) model the amount of mucus in the non-depleted wells was 3-, 8- and 4-fold higher than in the mucus-depleted wells of the Calu-3, SPOC1 and UNCN3T cells respectively. The permeability of 'high mucus' cells to testosterone was significantly less in SPOC1 and UNCN3T cells (P < 0.05) but not Calu-3 cells. Mucin secretion and cytokine release were investigated as indicators of drug irritancy in the SPOC1 and UNCN3T cell lines. A number of inhaled drugs significantly increased mucin secretion at high concentrations and the release of IL-6 and IL-8 from SPOC1 or UNCN3T cells (P < 0.05). SPOC1 and UNCN3T cell lines are better able to model the effect of mucus on drug absorption than the Calu-3 cell line and are proposed for use in assessing drug-mucus interactions in inhaled drug and formulation development.

The human bronchial epithelial cell line 16HBE14o- as a model system of the airways for studying drug transport.

The 16HBE14o- cell line, which forms polarised cell layers in vitro, provides a promising opportunity to develop a convenient epithelial cell culture model in which respiratory drug transport can be evaluated in vitro. This study investigated the effect of cell seeding density, collagen substratum and time in culture on the development of barrier properties in this cell line, after which the permeability of the 16HBE14o- cell layers to a series of solutes was studied. Seeding cells at a density of 2.5 x 10(5) cells per cm(2) on a monofibrillar Vitrogen-100 collagen substratum, followed by culture at an air-liquid interface for 6 days resulted in cell layers with a transepithelial electrical resistance (TER) of 247+/-47 omegacm(2) and an apparent permeability coefficient of 2.5 x 10(-6)cms(-1) for mannitol. The permeability of the 16HBE14o- cells to hydrophilic molecules (logP<1.9) was of an order of magnitude greater than that of typical alveolar cell cultures, possibly reflecting barrier properties more representative of the airways. More lipophilic drugs showed higher permeabilities indicating a sigmoidal relationship between permeability and lipophilicity similar to that observed for solute transport across primary cultured epithelial cell layers. These results indicate that under appropriate culture conditions, 16HBE14o- cell layers provide a discriminatory barrier to solute transport.

Toxicological evaluation of mixtures of nonionic surfactants, alone and in combination with oil.

The toxicity to human bronchial (16-HBE14o-) epithelium cells of nonionic surfactants, polyoxyethylene-10-oleyl ether (C(18:1)E(10)), polyoxyethylene-10-dodecyl ether (C(12)E(10)), and N,N-dimethyl-dodecylamine-N-oxide (C(12)AO) alone or in combination with a range of pharmaceutically acceptable oils (namely, ethyl esters and triglyceride oils), was determined with the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Regardless of the presence of oil, all C(12)E(10)- and C(12)AO-containing systems were toxic at concentrations around or below their critical aggregation concentrations (as determined by surface tension measurements), suggesting that surfactant toxicity was due to the disruption caused by the partitioning of monomeric surfactant into the cell membrane. Systems prepared from C(18:1)E(10) alone or in combination with a low-molecular-weight oil, such as ethyl butyrate or tributyrin, were toxic above their critical aggregation concentration. In contrast, systems prepared from C(18:1)E(10) in combination with a high-molecular-volume oil (e.g., ethyl oleate, Miglyol 812, or soybean oil) were toxic only at concentrations significantly greater than their critical aggregation concentration, suggesting that in these cases surfactant toxicity was mediated by the aggregated form of the surfactant solubilizing components of the cell membrane. In the C(18:1)E(10)-stabilized system, it is proposed that toxicity was significantly reduced on incorporation of high-molecular-volume oils because these oils cause formation of a distinct oil core in the aggregates that leads to a reduction in the ability of the system to solubilize components of the cell membrane.