High-Throughput Radiometric CYP2C19 Inhibition Assay Using Tritiated (<i>S</i>)-Mephenytoin

Marco, Annalise Di; Cellucci, Antonella; Chaudhary, Ashok; Fonsi, Massimiliano; Laufer, Ralph

doi:10.1124/dmd.107.016345

Cited by 14 publications

(8 citation statements)

References 27 publications

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“…Whereas almost linear dependence was estimated during the studied period for the lower concentration of CYP2C9, a slight nonlinearity was found at higher CYP2C9 concentration. Similar behaviour was recently observed by Di Marco et al [29] by means of a radiometric CYP2C9 assay. A range of CYP2C9 concentrations was tested as well.…”

Section: Resultssupporting

confidence: 78%

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Study of recombinant cytochrome P450 2C9 activity with diclofenac by MEKC

et al. 2007

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Cytochrome P450 2C9 (CYP2C9) is one of the most important isoforms in human liver involved in the metabolism of a large number of therapeutic agents. The aim of this paper is to demonstrate the applicability of CE for the determination of the enzymatic activity of CYP2C9 with diclofenac as a probe substrate. MEKC with SDS as a pseudostationary phase was used for this purpose. Compared to other assays, the MEKC-based method is rapid, can be automated and requires only a small quantity of enzymes and substrate. Moreover, the enzymatic reaction can be monitored with high sensitivity and repeatability even when the reaction mixture is used for the analysis without any pretreatment. The kinetic study on the given enzymatic reaction was also performed since the basic characterization of drug biotransformation generally begins with the enzyme kinetic analysis of metabolite formation. As a result, the Michaelis constant and maximum reaction velocity were evaluated, the values 3.44 +/- 0.45 microM and 19.78 +/- 0.76 nmol min(-1) nmol(-1), respectively, were in agreement with the literature data. On the other hand, a slight deviation from typical Michaelis-Menten kinetics with a weak positive cooperativity was found at diclofenac concentrations below 2 microM. The same atypical kinetic behavior of CYP2C9 was also observed by other authors.

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Section: Resultssupporting

confidence: 78%

“…As mentioned above, Di Marco et al [29] published recently a highly sensitive radiometric method for monitoring the CYP2C9 activity with diclofenac. This assay is based on the release of tritium as tritiated water from the diclofenac labelled with tritium in the 4 0 -position.…”

Section: Resultsmentioning

confidence: 96%

Study of recombinant cytochrome P450 2C9 activity with diclofenac by MEKC

et al. 2007

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“…Based on the results described herein, it is concluded that EE is not a submicromolar inhibitor of human P450s in vitro (Table 3). The lack of submicromolar inhibition of CYP2C19 Rodrigues and Lu, 2004;Di Marco et al, 2007), CYP2C8 (Walsky et al, 2005), CYP2C9 , CYP2B6 (Walsky et al, 2006), and CYP1A2 (Karjalainen et al, 2008) activity in vitro is consistent with the literature. To our knowledge, however, this is the first report describing the assessment of EE as an in vitro inhibitor of CYP2J2, CYP2D6, CYP1B1, and CYP1A1.…”

Section: Discussionsupporting

confidence: 79%

“…Other authors have reported IC 50 s for (S)-mephenytoin with HLM in the range of ϳ3.5 to 20 M, although no attempt was made to correct for microsomal binding (Rodrigues and Lu, 2004;Di Marco et al, 2007). To date, all the reported K i and IC 50 values for CYP2C19 are still considerably higher than the calculated total concentration of EE in enterocytes (ϳ6.0 nM) and hepatic portal vein (ϳ0.4 nM).…”

Section: Discussionmentioning

confidence: 86%

Further Assessment of 17α-Ethinyl Estradiol as an Inhibitor of Different Human Cytochrome P450 Forms in Vitro

Chang¹,

Chen²,

Yang³

et al. 2009

Drug Metab Dispos

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ABSTRACT:17␣-Ethinyl estradiol (EE) was systematically evaluated as a reversible and time-dependent inhibitor of 11 human drug-metabolizing cytochromes P450 (P450s) (CYP1A1, CYP1A2, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, and CYP3A5) in vitro. When ranked, the lowest IC 50 Reports continue to appear describing drug interactions involving oral contraceptive (OC) formulations containing 17␣-ethinyl estradiol (EE). For example, Hilli et al. (2008) reported recently that melatonin (MEL) area under the plasma concentration versus time curve (AUC) is increased (ϳ5-fold), and the 6-hydroxy melatonin (MEL 6-OH)/ MEL AUC ratio is decreased (88%). CYP1A2 is known to play a major role in the metabolism of MEL in human liver microsomes (HLM), so the authors deduced that the enzyme was the locus of the interaction (Facciolá et al., 2001;Härtter et al., 2001;Ma et al., 2005). EE-containing OCs exert a similar effect on the pharmacokinetics (PK) of tizanidine (Granfors et al., 2005). Both tizanidine and MEL are low oral bioavailability (Ͻ25%) CYP1A2 substrates because of first-pass metabolism (Härtter et al., 2001;Granfors et al., 2005). Although caffeine and theophylline also serve as CYP1A2 substrates, they undergo minimal first pass, are highly bioavailable, and the impact of EE is less marked (ϳ40% decrease in clearance) (Roberts et al., 1983;Balogh et al., 1995). Despite these clinical data, it is only very recently that assessment of CYP1A2 inhibition in vitro has been described previously (Karjalainen et al., 2008). In fact, Karjalainen et al. (2008) reported EE as a relatively weak inhibitor of phenacetin O-deethylation (POD) activity in HLM (low K m component; IC 50 ϭ 24 M).Drug interactions with EE-containing OC formulations have also included a number of CYP2C19 substrates, such as omeprazole, mephenytoin, and proguanil (Hägg et al., 2001; Rodrigues and Lu, 2004, references therein;Shelepova et al., 2005). Likewise, drug interactions with imipramine and selegiline have been described previously (Abernethy et al., 1984;Laine et al., 1999). The latter is a CYP2B6 and CYP2C19 substrate, also with a low oral bioavailability (Ͻ10%), and greater than 10-fold increases in AUC have been reported with OCs (Laine et al., 1999;Benetton et al., 2007). More importantly, the contribution of CYP2C19 after oral dosing of selegiline is thought to be minimal (Laine et al., 2001). Although CYP2B6 is implicated, there are no reports describing the impact of CYP2B6 genotype (or phenotype) on the oral PK of selegiline, and its contribution in vivo is not known. Moreover, the clinical drug interaction between EE and a CYP2B6 probe (bupropion) is not significant despite evidence for mechanism-based inhibition in vitro Palovaara et al., 2003). The same can also be said for the observed mechanism-based inhibition of CYP3A4 and CYP3A5 in vitro because the effect of EE on the PK of CYP3A substrates (e.g., midazolam and nifedipine) is minimal (Balogh et al., 1998; Article, publication date, and citation information c...

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“…The concentrations of the secondary amines in the inactivation assays and estimates of reversible affinities for their respective enzymes were as follows: DES ϭ 50 M with CYP2C11 (IC 50 ϭ 5 M, data not shown), (S)-FLX ϭ 10 M with CYP2C19 (IC 50 ϭ 4.7 M in HLMs) (Di Marco et al, 2007), and MA ϭ 2 M with CYP3A4 (K i ϭ 2 M in HLMs) (Zhao et al, 2007). The secondary hydroxylamine substrate concentrations used were lower to facilitate data acquisition and curve-fitting: DESOH ϭ 1 M, (S)-FLXOH ϭ 5 M, and MAOH ϭ 0.5 M. The initial rates of the loss of enzyme activity caused by the secondary amines and secondary hydroxylamines were determined in triplicate in the presence and absence of fixed concentrations of the primary amines at their respective K i or IC 50 values.…”

Section: Downloaded Frommentioning

confidence: 99%