Proteolysis of human calcitonin in excised bovine nasal mucosa: elucidation of the metabolic pathway by liquid secondary ionization mass spectrometry (LSIMS) and matrix assisted laser desorption ionization mass spectrometry (MALDI).

PURPOSE: Two calcitonins, i.e. human calcitonin (hCT) and, for comparison, salmon calcitonin (sCT), were chosen as peptide models to investigate nasal mucosal metabolism. METHODS: The susceptibility of hCT and sCT to nasal mucosal enzymes was assessed by in-and-out reflection kinetics experiments in an in vitro model based on the use of freshly excised bovine nasal mucosa, with the mucosal surface of the mucosa facing the peptide solution. The kinetics of CT degradation in the bulk solution was monitored by HPLC. Peptide sequences of the main nasal metabolites of hCT were analyzed by using both liquid secondary ionization mass spectrometry (LSIMS), following HPLC fractionation of the metabolites, and matrix-assisted laser desorption ionization mass (MALDI) spectrometry. For sCT, the molecular weights of two major metabolites were determined by LC-MS with electrospray ionization. RESULTS: Both CTs were readily metabolized by nasal mucosal enzymes. In the concentration range studied metabolic rates were higher with hCT than with sCT. Presence of endopeptidase activities in the nasal mucosa was crucial, cleaving both calcitonins in the central domain of the molecules. CONCLUSIONS: Typically, initial metabolic cleavage of hCT in nasal mucosa is due to both chymotryptic- and tryptic-like endopeptidases. The subsequent metabolic break-down follows the sequential pattern of aminopeptidase activity. Tryptic endopeptidase activity is characteristic of nasal sCT cleavage.

Evaluation of the human cell line RPMI 2650 as an in vitro nasal model.

We have investigated a human nasal cell line, RPMI 2650 (ATCC), as an in vitro model to assess the absorption and tolerability of nasally administrated peptides. This cell line was cultured on different filters (i.e. Nunc, Costar, Falcon, Millipore, and Becton) which were uncoated or coated with collagen types I or IV, laminin, fibronectin and extracellular matrix. Cell line morphology, capability of forming a cell layer and phenotype were analyzed by light-, fluorescence- and electron microscopy. The morphological analysis showed that RPMI 2650 cells were forming cell clusters on one group of filters (i.e., Nunc, Costar, Falcon and Becton filters coated with collagen types I or IV, laminin and extracellular matrix). On the second group of filters (i.e., Becton and Millipore filters, coated with fibronectin) the cells had the tendency to spread in a cell layer. In both groups of filters, the cells never showed cell polarization, nor microvilli and tight junctions. Phenotyping of this cell line was performed by indirect immunofluorescence using monoclonal antibodies against human cytokeratins 10, 17 and 18 (markers of epithelial cells), desmin (markers of mesothelial cells) and vimentin (marker of mesothelial cells). Vimentin was strongly expressed, cytokeratins 10, 17 and 18 were weakly expressed, and desmin was not expressed. The human nasal cell line RPMI 2650 was not able to form a tight cell layer under these cell culture conditions. The limits of this cell line as an in vitro nasal model for drug absorption is discussed.

Tolerability and absorption enhancement of intranasally administered octreotide by sodium taurodihydrofusidate in healthy subjects.

Nasal sprays containing different concentrations of the somatostatin analogue octreotide and sodium tauro-24,25-dihydrofusidate (STDHF) as an absorption promoter were evaluated in two consecutive pharmacokinetic studies in healthy volunteers to characterize their bioavailability and local tolerability. The concentrations of STDHF were selected on the basis of a phase diagram generated by a dynamic laser light-scattering technique to ensure that the mixture was above the critical micellar concentrations. Compared to a 50-micrograms subcutaneous injection, the nasal spray formulation without STDHF had a mean relative bioavailability of 17.9%. For nasal formulations containing 3 and 1.65% (w/v) of STDHF, the bioavailability increased to 29.0 and 25.7%, respectively. The enhancement of nasal absorption was dependent on the STDHF concentrations as shown by decreasing the amounts to 1.2 and 0.8% (w/v) for tolerability reasons; the bioavailability was reduced to 15.3 and 20.5% in these cases, respectively. The local tolerability of all STDHF-containing sprays was poor, leading to stinging sensations and lacrimation. The poor local tolerability of the octreotide nasal spray containing different concentrations of STDHF required for effective nasal absorption enhancement appears to be impractical for further clinical development. These findings clearly stress the necessity to investigate tolerability and safety issues of new drug delivery systems in early developmental phases.