Experimental condition-related variables, such as seeding density, culture matrix and medium, start and duration of treatment, affecting the response of plateable thawed cryopreserved human hepatocytes to cytochrome P450 inducers can be reduced by optimizing critical steps of the protocols.

ABSTRACT:Benzyloxyresorufin-O-dealkylation (BROD) is usually used as a marker of cytochrome P450 (P450) 2B1 in rat. However, some reports show that CYP1A2 is also highly implicated. The purpose of the present study was to establish bupropion (BUP) hydroxylation, but not BROD, as a selective in vitro marker of CYP2B1 catalytic activity. IC 50 for BROD and BUP hydroxylation were equivalent (40.8 ؎ 4.6 and 41.8 ؎ 3.4 M, respectively) when using liver microsomes from ␤-naphthoflavone-pretreated rats in the presence of metyrapone (CYP2B1 inhibitor). When using the same microsomes in the presence of CYP1A1/2-selective inhibitor ␣-naphthoflavone, we found an IC 50 of 2.5 ؋ 10 ؊3 ؎ 0.8 ؋ 10 ؊3 M for BROD and >100 M for BUP hydroxylation. These results suggest that CYP2B1 is similarly involved in both activities, whereas CYP1A2 is involved in BROD activity but not in BUP hydroxylation. BUP hydroxylation was assessed in microsomes from baculovirus-infected insect cells coexpressing NADPH-P450 oxidoreductase, and 14 rat P450s and kinetic parameters (K m and V max ) were determined. BUP hydroxylation was predominantly catalyzed by CYP2B1 (75% of total hydroxybupropion formation), low activity was detected with CYP2E1 and CYP2C11 (10.9 and 8.7% of total hydroxybupropion, respectively), and activity was almost undetectable with the other P450 isoforms at saturating substrate concentrations (2500 M), thereby validating the use of BUP as a diagnostic in vitro marker of CYP2B1 catalytic activity in rat.

1.  It has previously been demonstrated that metabolism of drugs via a single enzymatic pathway, particularly CYP3A4, is associated with increased risk for drug-drug interactions (DDI). Quantitative experimental systems as well as integrated prediction models to assess such risk during the preclinical phase are highly warranted. 2.  The present study was designed to systematically investigate the performance of human cryopreserved hepatocytes in suspension to predict fraction metabolized via CYP3A (fmCYP3A) by assessing the ketoconazole sensitive intrinsic clearance (CLint) for five prototypical CYP3A substrates with varying degree of CYP3A dependent CLint in twelve individual hepatocyte batches. 3.  We demonstrate that in contrast to well predicted mean hepatic metabolic clearance (CLH) and mean fmCYP3A data, the variability in CYP3A contribution for compounds having multiple metabolic pathways cannot be predicted from inhibition experiments using ketoconazole as inhibitor. Instead, data in the present paper indicate that the variability is larger after inhibition of CYP3A for compounds having multiple metabolic pathways. 4.  It is therefore recommended to estimate the average CLint and fmCYP3A for a given test compound in a series (n = 10) of individual human hepatocyte batches.

Characterization of glycosaminoglycans poses a challenge for current analytical techniques, as they are highly acidic, polydisperse and heterogeneous compounds. The purpose of this study is the separation and analysis of a partially depolymerized heparin-like glycosaminoglycan by on-line ion-pairing reversed-phase high-performance liquid chromatography/electrospray mass spectrometry. The gas-phase behavior of two synthesized glycosaminoglycans has been investigated. Dibutylamine was found to be the best suited ion-pairing reagents for mass spectrometry analysis. The optimized ion-pairing conditions provide reproducible and easily interpretable electrospray mass spectra in both negative and positive ESI modes. The glycosaminoglycans are detected as a non-covalent complex with amines. In fact, the observed ionic species and their gas-phase dissociation under CID conditions revealed the presence of salt bridge interactions in the gas phase.

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