Application of a generic fast gradient liquid chromatography tandem mass spectrometry method for the analysis of cytochrome P450 probe substrates

Ayrton, John; Plumb, Robert S.; Leavens, William J.; Mallett, David N.; Dickins, Maurice; Dear, Gordon J.

doi:10.1002/(sici)1097-0231(19980314)12:5<217::aid-rcm146>3.0.co;2-i

Cited by 97 publications

(85 citation statements)

References 3 publications

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“…3 The LC/MSbased assay utilizes conventional drug probes that are approved medicinal agents and that are CYPenzyme specific; therefore, more and more industrial laboratories are increasingly switching to this method. 7,8 Probe substrates must be selective, specific, and sensitive toward the CYP of interest. Over the years, pharmaceutical scientists have used numerous probe reactions for the various P450 enzymes to evaluate the inhibitory potential of a new drug.…”

Section: Introductionmentioning

confidence: 99%

In vitro assessment of cytochrome P450 inhibition: Strategies for increasing LC/MS‐based assay throughput using a one‐point IC50 method and multiplexing high‐performance liquid chromatography

Tong¹,

Pan

Mordenti³

et al. 2007

Journal of Pharmaceutical Sciences

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Section: Introductionmentioning

confidence: 99%

In vitro assessment of cytochrome P450 inhibition: Strategies for increasing LC/MS‐based assay throughput using a one‐point IC50 method and multiplexing high‐performance liquid chromatography

Tong¹,

Pan

Mordenti³

et al. 2007

Journal of Pharmaceutical Sciences

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“…The now routine access to recombinant P450s and HLMs have made these in vitro tools highly valuable for determining the extent and route of the metabolism of new chemical entities and in screening for inhibitors of drug-metabolizing enzymes (Ayrton et al, 1998;Moody et al, 1999;McGinnity et al, 2000).…”

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confidence: 99%

Evaluation of Fresh and Cryopreserved Hepatocytes as in Vitro Drug Metabolism Tools for the Prediction of Metabolic Clearance

McGinnity

Soars²,

Urbanowicz³

et al. 2004

Drug Metab Dispos

259

191

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ABSTRACT:The intrinsic clearances (CL int ) of 50 neutral and basic marketed drugs were determined in fresh human hepatocytes and the data used to predict human in vivo hepatic metabolic clearance (CL met ). A statistically significant correlation between scaled CL met and actual CL met was observed (r 2 ‫؍‬ 0.48, p < 0.05), and for 73% of the drugs studied, scaled clearances were within 2-fold of the actual clearance. These data have shown that CL int data generated in human hepatocytes can be used to provide estimates of human hepatic CL met for both phase I and phase II processes. In addition, the utility of commercial and in-house cryopreserved hepatocytes was assessed by comparing with data derived from fresh cells. A set of 14 drugs metabolized by the major human cytochromes P450 (P450s) (CYP1A2, 2C9, 2C19, 2D6, and 3A4) and uridine diphosphate glucuronosyltransferases (UGT1A1, 1A4, 1A9, and 2B7) have been used to characterize the activity of freshly isolated and cryopreserved human and dog hepatocytes. The cryopreserved human and dog cells retained on average 94% and 81%, respectively, of the CL int determined in fresh cells. Cryopreserved hepatocytes retain their full activity for more than 1 year in liquid N 2 and are thus a flexible resource of hepatocytes for in vitro assays. In summary, this laboratory has successfully cryopreserved human and dog hepatocytes as assessed by the turnover of prototypic P450 and UGT substrates, and both fresh and cryopreserved human hepatocytes may be used for the prediction of human hepatic CL met .

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“…Availability of good quality fresh liver tissue limits human hepatocyte experiments, but, increasingly, cryopreservation technology is allowing hepatocytes to become an "off-the-shelf" reagent (Li, 1999). The end-points used for P450 inhibition screening in the pharmaceutical environment are typically one of the following; liquid scintillation counting of radioactivity liberated during site-specific metabolism (Moody et al, 1999), selective analysis of fluorescent metabolites (Crespi et al, 1998), and mass spectrometry (Ayrton et al, 1998;Weaver et al, 2003). In drug discovery, assays are routinely conducted at the K m for the substrate since, under these conditions, for most inhibitors, K i ϭ IC 50 /2 or IC 50 .…”

mentioning

confidence: 99%

Prediction of Cyp2c9-Mediated Drug-Drug Interactions: A Comparison Using Data From Recombinant Enzymes and Human Hepatocytes

McGinnity

Tucker²,

Trigg³

et al. 2005

Drug Metab Dispos

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ABSTRACT:The IC 50 values of 14 drugs were determined in recombinantly expressed CYP2C9 (rCYP2C9) and human hepatocytes and the data used to simulate clinical area under the plasma concentration-time curve (AUC) changes upon coadministration with prototypic CYP2C9 substrates. There was an excellent correlation between IC 50 Up to 2.8% of hospital admissions may be a consequence of drug-drug interactions (DDIs) (Jankel and Fitterman, 1993). Although metabolic DDIs have been reported for several enzyme families, it is clearly recognized that inhibition of cytochrome P450 (P450)-dependent metabolism is one of the more prevalent sources of such reactions and may have a serious outcome (Bertz and Granneman, 1997). Experiences with terfenadine (Honig et al., 1993), cisapride (Ahmad and Wolfe, 1995), and mibefradil (Krayenbuhl et al., 1999) have highlighted the importance of understanding the enzymology of drug metabolism to assess its impact on pharmacodynamics and toxicology.An assessment of the potential of a new chemical entity to cause a DDI via inhibition of P450 metabolism is important early in the drug discovery process. Thus, a battery of automated in vitro screens to determine the degree of P450 inhibition are now routinely used in drug metabolism and pharmacokinetics departments across the pharmaceutical industry. Such screens are used both for the evaluation and optimization of potential candidate drugs and for prioritizing and designing suitable in vitro and clinical interaction studies in the drug development phase. The ability to predict in vivo DDIs from human in vitro assays would represent a major advance in drug discovery.Approaches to predicting P450-mediated DDI potential have traditionally involved the use of human liver microsomes, although more recently, the use of recombinant cytochrome P450s (rP450s) has become widespread. More often than not, these assays focus on the five major drug-metabolizing P450s: CYP1A2, 2C9, 2C19, 2D6, and 3A4. Human hepatocytes in primary culture provide the closest in vitro model to human liver and have been excellent tools for elucidating the metabolic profile of drugs, for predicting hepatic metabolic clearance (McGinnity et al., 2004;Riley et al., 2005), for studying hepatotoxicity of drugs (Gomez-Lechon et al., 2001), and for the mechanistic understanding of DDIs mediated via P450 induction (LeCluyse et al., 2000). There are sporadic reports of the use of human hepatocytes to characterize P450 inhibition Cohen et al., 2000;Oleson et al., 2004;Zhao et al., 2005), but it is notable that investigations of inhibitory mechanisms remain under-represented compared with studies on metabolism and induction. This is likely due to the relative accessibility of recombinant P450s and their overall applicability in predicting clinically relevant DDIs. Availability of

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Application of a generic fast gradient liquid chromatography tandem mass spectrometry method for the analysis of cytochrome P450 probe substrates

Cited by 97 publications

References 3 publications

In vitro assessment of cytochrome P450 inhibition: Strategies for increasing LC/MS‐based assay throughput using a one‐point IC50 method and multiplexing high‐performance liquid chromatography

In vitro assessment of cytochrome P450 inhibition: Strategies for increasing LC/MS‐based assay throughput using a one‐point IC50 method and multiplexing high‐performance liquid chromatography

Evaluation of Fresh and Cryopreserved Hepatocytes as in Vitro Drug Metabolism Tools for the Prediction of Metabolic Clearance

Prediction of Cyp2c9-Mediated Drug-Drug Interactions: A Comparison Using Data From Recombinant Enzymes and Human Hepatocytes

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