Development of analytical methods for the quantification of metabolites of lesogaberan in a MIST investigation.

Analytical methods were developed for the determination of six metabolites of lesogaberan to be used in quantitative determinations of metabolites according to the guidelines of Metabolites in Safety Testing. The γ-amino butyric acid type B receptor agonist lesogaberan and its metabolites are small polar molecules and hydrophilic interaction liquid chromatography was found to be a suitable separation mode. The samples were prepared using protein precipitation and negative electrospray ionization tandem mass spectrometry was used for detection. Initially, exploratory methods for six metabolites were set up for analysis of human plasma samples taken after repeated administration of a high oral dose of lesogaberan. The purpose was to establish which metabolites were present at concentrations significant for further investigation. Four of the six metabolites were then found at clearly detectable concentrations. The analytical methods for these four metabolites were further elaborated and then taken through a qualification procedure, which showed acceptable accuracy (86-114%), precision (<9%) and good linearity in the range 0.03-5 µmol/L. No interferences were seen from endogenous plasma components.

Effects of ketoconazole on the in vivo biotransformation and hepatobiliary transport of the thrombin inhibitor AZD0837 in pigs.

Ketoconazole has been shown in clinical trials to increase the plasma exposure of the oral anticoagulant prodrug AZD0837 [(2S)-N-{4- [(Z)-amino(methoxyimino)methyl]benzyl}-1-{(2R)-2-[3-chloro-5-(difluoromethoxy)phenyl]-2-hydroxyethanoyl}-azetidine-2-carboxamide] and its active metabolite, AR-H067637 [(2S)-N-{4-[amino(imino)methyl]benzyl}-1-{(2R)-2-[3-chloro-5-(difluoromethoxy)phenyl]-2-hydroxyethanoyl}-azetidine-2-carboxamide]. To investigate the biotransformation of AZD0837 and the effect of ketoconazole on this process, we used an experimental model in pigs that allows repeated sampling from three blood vessels, the bile duct, and a perfused intestinal segment. The pigs received AZD0837 (500 mg) given enterally either alone (n = 5) or together with single-dose ketoconazole (600 mg) (n = 6). The prodrug (n = 2) and its active metabolite (n = 2) were also administered intravenously to provide reference doses. The plasma data revealed considerable interindividual variation in the exposure of the prodrug, intermediate metabolite, and active metabolite. However, AR-H067637 was detected at very high concentrations in the bile with low variability (Ae(bile) = 53 ± 6% of the enteral dose), showing that the compound had indeed been formed in all of the animals and efficiently transported into the bile canaliculi. Concomitant dosing with ketoconazole increased the area under the plasma concentration-time curve for AZD0837 (by 99%) and for AR-H067637 (by 51%). The effect on the prodrug most likely arose from inhibited CYP3A-mediated metabolism. Reduced metabolism also seemed to explain the increased plasma exposure of the active compound because ketoconazole prolonged the terminal half-life with no apparent effect on the extensive biliary excretion and biliary clearance. These in vivo results were supported by in vitro depletion experiments for AR-H067637 in pig liver microsomes with and without the addition of ketoconazole.