Influence de l'inflammation sur le metabolisme catalyse par les cytochromes P450 chez le rat. Approches in vitro et implications sur la pharmacocinetique d'agonistes delta (French text).

摘要

This research program was undertaken to give a representation of the multidisciplinarity to which drug metabolism and pharmacokinetic (DMPK) scientists are exposed in the pharmaceutical industry. We focused principally on in vitro systems and hepatic cytochromes P450 (CYPs); which represent major determinants of the elimination of several molecules in rats and humans. In the first section of this thesis, studies were performed to identify the CYPs involved in morphine-N-demethylation in human liver microsomes.; As a first step, a simple, rapid and sensitive HPLC-MS method that allowed the quantification of morphine and its principal metabolites in various biological matrixes was developed and validated. Traditional in vitro CYP phenotyping studies were then performed. Our in vitro findings confirmed that hepatic CYP3A4, and to a lesser extent CYP2C8, play an important role in morphine-N-demethylation in man.; Focusing on analgesics and pain, the goals of the second part of this thesis were to characterize an inflammatory pain model and its effect on DMPK and evaluate the in vitro pharmacology and in vivo pharmacokinetics of non-peptidic delta-receptor agonists and their main metabolites. In the rat, a single Freund's Complete Adjuvant (FCA) injection in the hind paw lead to the development of an inflammatory response with increased alpha-1-glycoprotein (AGP) and IL-6 plasma levels in the early days that follow injection. These changes were accompanied by a rapid decrease in total CYP contents and the selective downregulation of CYP2B, CYP2C6, CYP2C11 and CYP2E1 in FCA-treated rat liver microsomes (RLM). Our work corroborated the major role of IL-6 in eliciting these changes. In rats, we demonstrated that SNC80 is mainly eliminated by CYP-mediated hepatic metabolism and is highly bound to plasma proteins (>94%) and AGP. Although its absorption was essentially complete after oral administration, this delta-agonist presented poor oral bioavailability (4%) because of extensive pre-systemic metabolism. We showed that, using SNC80, the biochemical modifications observed in the FCA-treated rats lead to the decrease of its free fraction in plasma (∼30%) and of its intrinsic clearance in the RLM (∼40%). These modifications led to a decrease (22%) in the plasma clearance of SNC80 in vivo. In contrast, our results did not show any modifications of the oral bioavailability of SNC80 in FCA-treated rats. We suggest that FCA-induced non-hepatic mechanisms may have compensated for the decrease in plasma clearance, preventing an increase in the oral bioavailability. The last part of the thesis showed that ARM390 is a highly selective delta-agonist with moderate potency in vitro. Its unique selectivity, good oral bioavailability (70%) and the poor in vitro activity of its main metabolite (ARM827) make this delta-agonist one of the most attractive tools currently available. One should exercise caution when using (+/-)-BW373U86 or SNC80 as pharmacological tools in vivo as they either lack selectivity or release pharmacologically active metabolite(s) that could contribute to pharmacological activity.; Overall, the work carried out in this thesis highlights the importance of DMPK and in vitro systems in many aspects of clinical and pre-clinical pain research.