Characterization of the metabolites of alosetron in experimental animals and human

Abstract

The metabolism of radiolabelled alosetron was studied in rat, dog, rabbit, mouse and human. The metabolism in rat and dog was studied at a low and an elevated dose designed to generate sufficient quantities of metabolite for definitive identification. A strategy for the characterization of metabolites in cases of extensive metabolism was developed and demonstrated for alosetron. Semi-preparative high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) and liquid chromatography-nuclear magnetic resonance (HPLC-NMR) enabled the isolation and characterization of 28 metabolites of alosetron. The characterization of the metabolites in animal excreta facilitated the identification of human systemic metabolites.

The metabolism of the 5HT3antagonists ondansetron, alosetron and GR87442 I: A comparison ofin vitroandin vivometabolism andin vitroenzyme kinetics in rat, dog and human hepatocytes, microsomes and recombinant human enzymes

“…Quantitative differences in the production of metabolites of the 5HT 3 antagonists were apparent between in vitro systems. Neither microsomes nor hepatocytes completely reflected the quantitative in vivo metabolite profile of the 5HT 3 antagonists as previously reported in pre-clinical species or human (Wolf 2000;Koch et al 2004;Ismail et al 2005). In general, the production of more lipophilic, CYP3A4 mediated metabolites, especially carbonyl metabolites, predominated in microsomes compared with hepatocytes.…”

“…*metabolites previously identified in human liver micosomes. metabolites due to multiple sequential oxidation of alosetron previously reported in animals and human (Ismail et al 2005) were not observed as major metabolites in rat and dog microsomes or hepatocytes. A number of minor mono-oxygenated metabolites of alosetron were detected but were not given definitive structural assignments owing to lack of fragmentation information.…”

“…However, there is more extensive modification of the imidazole ring system of alosetron compared with that reported for ondansetron. Owing to the need for increased analytical sensitivity further metabolites of alosetron, such as pyridoindole N-hydroxymethyl alosetron, have been assigned more recently (Ismail et al 2005). The N-methyl hydroxylation of xenobiotics has previously been reported (Ross et al, 1981, although these metabolites are generally thought to be an unstable intermediate in the formation of N-desmethyl metabolites (Parkinson 1986).…”

“…Conversely, with alosetron, the linkage is via a carbon atom in the imidazole group and nitrogen atom on the pyridoindole tricyclic ring. Alosetron is transformed into a large number of phase I and sequential phase II metabolites in pre-clinical species and man, with no single metabolite occurring at levels greater than 15% of the dose (Ismail et al 2005). Metabolically vulnerable sites exist on both the tricyclic pyrido-indol nucleus and the imidazole group of alosetron, and metabolism is primarily CYP-mediated (Koch et al 2004).…”

Analytical Tools and Approaches for Metabolite Identification in Early Drug Discovery

“…Analytical (4.6 mm) columns can support larger injections but also dilute the eluted sample as presented to the NMR detection region. The combination of HPLC and online SPE (70,71) for concentration of dilute samples prior to introduction to a capillary or cryogenic (72) probe NMR appears the best current alternative to full (73) or partial (74) offline isolation. In a very rare case, X-ray crystallography (75) can be used to exactly define metabolite stereochemistry in systems where even NMR fails to define all atom positions.…”