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Pre-systemic elimination by local enzymatic degradation can play a key role in limiting the bioavailability of intranasally administered drugs. Despite remarkable advancement in the characterization of the nasal biotransformative enzymes, knowledge of the role of the nasal mucosa in limiting bioavailability of therapeutic agents is still inadequate. The aim of this work was to evaluate the expression and substrate biotransformation activity of cytochrome P450 enzymes in the nasal mucosa using bovine olfactory and respiratory explants as in vitro models.
Gene expression and localization of major CYP450 isoforms in the nasal mucosa were examined using RT-PCR and immunohistochemistry. The bovine nasal mucosa showed abundant expression of CYP2A6 and 3A4 genes whereas 1A1, 1A2, 2C9, and 2C19 isoforms were expressed at much lower levels. The CYP450 proteins were observed to be present in the epithelial layer and in submucosal glandular cells.
The diffusion of melatonin, a CYP1A2 substrate, and the appearance of 6-hydroxymelatonin, its primary metabolite, across bovine olfactory and respiratory explants was measured, and nasal olfactory and respiratory microsomal preparations were used to quantify the kinetic parameters for melatonin 6-hydroxylation. Results indicated that bovine olfactory and respiratory CYP450 isoforms were metabolically active towards melatonin metabolism, and the respiratory mucosa demonstrated the greatest melatonin 6-hydroxylation activity.
Numerical simulations were used to probe the effects of the relative magnitudes of the permeability coefficient and enzymatic parameters on net substrate mass transfer across nasal mucosal tissues. The simulations indicated that the concentration gradient of the drug coupled with its permeability coefficient were the most significant factors controlling the transport of drugs across the mucosal tissue. Enzymatic degradation decreased the flux of drugs across the mucosa and had the greatest impact on low permeability compounds.
The results from these studies show that the bovine nasal mucosa possesses significant metabolic activity, and the flux of a metabolically labile substrate across the nasal mucosa can be significantly reduced by its enzymatic degradation within the tissue. Use of kinetic modeling to characterize of the extent of biotransformation in the nasal mucosa enables the identification of metabolism-limited bioavailability of intranasally administered drug compounds.