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Research ArticleArticle

IMPACT OF INCUBATION CONDITIONS ON BUFURALOL HUMAN CLEARANCE PREDICTIONS: ENZYME LABILITY AND NONSPECIFIC BINDING

Robert S. Foti and Michael B. Fisher
Drug Metabolism and Disposition March 2004, 32 (3) 295-304; DOI: https://doi.org/10.1124/dmd.32.3.295
Robert S. Foti
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Michael B. Fisher
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Abstract

Human liver microsomes (HLMs) are frequently utilized in drug discovery to predict the human clearance of a compound. The extent to which the incubation conditions affect the accuracy of a human clearance prediction was determined for bufuralol. HLMs were preincubated at 37°C for varying times (5–120 min) with and without NADPH, and the remaining enzyme activity was determined by incubating compounds that have been characterized to be selective for individual cytochromes P450 or flavin-containing monooxygenase 3. CYP2D6, the high-affinity component of bufuralol metabolism, was shown to be the least stable of the isoforms studied. The loss of CYP2D6 activity was further examined by determining the kinetics of 1′-hydroxybufuralol formation after different preincubation time periods, by using reactive oxygen species (ROS) scavengers, and by utilizing Western blotting techniques. A 3-fold decrease in Vmax was observed over 2 h, whereas the Km remained constant. ROS scavengers were able to block enzyme lability, and Western blots revealed no apparent loss of immunoreactive enzyme. The protein binding of bufuralol was determined in HLMs, recombinant CYP2D6, and human plasma. A prediction of theoretical bufuralol concentrations over a 120-min incubation that incorporated enzyme lability was performed and shown to be closer to actual data than if enzyme lability were ignored. Finally, a similar prediction using literature bufuralol data, coupled with the observed protein binding data, was used to illustrate that the most accurate predictions of bufuralol clearance are obtained when the amount of protein in the incubation is kept to a minimum and the overall incubation time is less than 20 min.

Footnotes

  • ↵1 Abbreviations used are: ADME, absorption, distribution, metabolism, and elimination; HLM, human liver microsome; ROS, reactive oxygen species; CYP2D6, cytochrome P450 2D6; CLint, microsomal intrinsic clearance; SOD, superoxide dismutase; CAT, catalase; DEF, deferoxamine; MRM, multiple reaction monitoring; HPLC, high performance liquid chromatography; CLp, predicted in vivo clearance; fu-plasma, fraction unbound in plasma; fu-mic, fraction unbound in microsomes; P450, cytochrome P450; fu-2D6, fraction unbound in recombinant CYP2D6; FMO, flavin-containing monooxygenase; rCYP2D6, recombinant CYP2D6.

    • Received September 9, 2003.
    • Accepted November 25, 2003.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 32 (3)
Drug Metabolism and Disposition
Vol. 32, Issue 3
1 Mar 2004
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Research ArticleArticle

IMPACT OF INCUBATION CONDITIONS ON BUFURALOL HUMAN CLEARANCE PREDICTIONS: ENZYME LABILITY AND NONSPECIFIC BINDING

Robert S. Foti and Michael B. Fisher
Drug Metabolism and Disposition March 1, 2004, 32 (3) 295-304; DOI: https://doi.org/10.1124/dmd.32.3.295

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Research ArticleArticle

IMPACT OF INCUBATION CONDITIONS ON BUFURALOL HUMAN CLEARANCE PREDICTIONS: ENZYME LABILITY AND NONSPECIFIC BINDING

Robert S. Foti and Michael B. Fisher
Drug Metabolism and Disposition March 1, 2004, 32 (3) 295-304; DOI: https://doi.org/10.1124/dmd.32.3.295
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