Mechanism-Based Inactivation of Cytochrome P450 3A4 by Mibefradil through Heme Destruction

Foti, R.; Rock, Dan A.; Pearson, Josh T.; Wahlstrom, Jan L.; Wienkers, Larry C.

doi:10.1124/dmd.111.038505

Cited by 39 publications

(38 citation statements)

References 34 publications

(33 reference statements)

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“…The enzyme structure is perturbed because it is incapable of forming a reduced CO-difference spectrum, indicating that the heme thiolate is disrupted. However, CYP3cide does not act by forming a metabolite-intermediate complex like other amines, and it does not act by forming an adduct to the porphyrin (Foti et al, 2011). Efforts to show covalent binding to the protein have been unsuccessful, although studies to detect drug-topolypeptide or drug to heme via LC/MS will continue to be an area of effort in our laboratories.…”

Section: Discussionmentioning

confidence: 99%

Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs

et al. 2012

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CYP3cide (PF-4981517; 1-methyl-3-[1-methyl-5-(4-methylphenyl)-1H-pyrazol-4-yl]-4-[(3S)-3-piperidin-1-ylpyrrolidin-1-yl]-1H-pyrazolo[3,4-d]pyrimidine) is a potent, efficient, and specific time-dependent inactivator of human CYP3A4. When investigating its inhibitory properties, an extreme metabolic inactivation efficiency (k inact /K I ) of 3300 to 3800 ml ⅐ min ؊1 ⅐ mol ؊1 was observed using human liver microsomes from donors of nonfunctioning CYP3A5 (CYP3A5 *3/*3). This observed efficiency equated to an apparent K I between 420 and 480 nM with a maximal inactivation rate (k inact ) equal to 1.6 min ؊1 . Similar results were achieved with testosterone, another CYP3A substrate, and other sources of the CYP3A4 enzyme. To further illustrate the abilities of CYP3cide, its partition ratio of inactivation was determined with recombinant CYP3A4. These studies produced a partition ratio approaching unity, thus underscoring the inactivation capacity of CYP3cide. WhenCYP3cide was tested at a concentration and preincubation time to completely inhibit CYP3A4 in a library of genotyped polymorphic CYP3A5 microsomes, the correlation of the remaining midazolam 1-hydroxylase activity to CYP3A5 abundance was significant (R 2 value equal to 0.51, p value of <0.0001). The work presented here supports these findings by fully characterizing the inhibitory properties and exploring CYP3cide's mechanism of action. To aid the researcher, multiple commercially available sources of CYP3cide were established, and a protocol was developed to quantitatively determine CYP3A4 contribution to the metabolism of an investigational compound. Through the establishment of this protocol and the evidence provided here, we believe that CYP3cide is a very useful tool for understanding the relative roles of CYP3A4 versus CYP3A5 and the impact of CYP3A5 genetic polymorphism on a compound's pharmacokinetics.

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Section: Discussionmentioning

confidence: 99%

Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs

et al. 2012

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“…25,26 Mibefradil (8) is an inhibitor of T-type Ca 2+ channels, 27 and was briefly marketed for migraine, but was withdrawn because of drug-drug interactions due to its potent inhibition of CYP3A4. 28 In an earlier study 29 it was shown to have no effect on signal-and extracellular Ca 2+ -dependent increases in [Ca 2+ ] stimulated by oxytocin. In our assay both 7 and 8 were active (albeit not very potent; IC 50 s 67 and above 100 µM respectively) against Orai1-mediated Ca 2+ influx (Table 1) but were not selective with respect to inhibition of the Ca V 2.2 channel (data not shown).…”

Section: Biologymentioning

confidence: 99%

Evaluation of known and novel inhibitors of Orai1-mediated store operated Ca 2+ entry in MDA-MB-231 breast cancer cells using a Fluorescence Imaging Plate Reader assay

Azimi

Flanagan

Stevenson

et al. 2017

Bioorganic & Medicinal Chemistry

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“…3, A and B) suggest that these kinetic schemes will result in log-linear PRA plots. However, there are many examples in the literature of curved PRA plots (He et al, 1998;Voorman et al, 1998;Kanamitsu et al, 2000;Yamano et al, 2001;Heydari et al, 2004;Obach et al, 2007;Bui et al, 2008;Foti et al, 2011). Both quasi-irreversible and partial inactivation kinetics result in concave upward plots (Figs.…”

mentioning

confidence: 99%

A Numerical Method for Analysis of In Vitro Time-Dependent Inhibition Data. Part 1. Theoretical Considerations

Nagar¹,

Jones²,

Korzekwa³

2014

Drug Metab Dispos

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Inhibition of cytochromes P450 by time-dependent inhibitors (TDI) is a major cause of clinical drug-drug interactions. It is often difficult to predict in vivo drug interactions based on in vitro TDI data. In part 1 of these manuscripts, we describe a numerical method that can directly estimate TDI parameters for a number of kinetic schemes. Datasets were simulated for Michaelis-Menten (MM) and several atypical kinetic schemes. Ordinary differential equations were solved directly to parameterize kinetic constants. For MM kinetics, much better estimates of K I can be obtained with the numerical method, and even IC 50 shift data can provide meaningful estimates of TDI kinetic parameters. The standard replot method can be modified to fit non-MM data, but normal experimental error precludes this approach. Non-MM kinetic schemes can be easily incorporated into the numerical method, and the numerical method consistently predicts the correct model at errors of 10% or less. Quasi-irreversible inactivation and partial inactivation can be modeled easily with the numerical method. The utility of the numerical method for the analyses of experimental TDI data is provided in our companion manuscript in this issue of Drug Metabolism and Disposition (Korzekwa et al., 2014b).

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Mechanism-Based Inactivation of Cytochrome P450 3A4 by Mibefradil through Heme Destruction

Cited by 39 publications

References 34 publications

Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs

Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs

Evaluation of known and novel inhibitors of Orai1-mediated store operated Ca 2+ entry in MDA-MB-231 breast cancer cells using a Fluorescence Imaging Plate Reader assay

A Numerical Method for Analysis of In Vitro Time-Dependent Inhibition Data. Part 1. Theoretical Considerations

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