Effect of ion suppression on judgment of enzyme inhibition and avoidance of error by utilizing a stable isotope-labeled probe substrate: example of CYP3A4 inhibition with [13C4,15N] labeled midazolam as a substrate.
An advantage of using LC-MS(/MS) for in vitro CYP inhibition screening is that it does not require extensive sample preparation and chromatographic separation. Attention must be paid, however, to ion suppression effects on analytes caused by the test compound as well as endogenous compounds. In this study, we have shown the ion suppression of 1'-hydroxymidazolam (analyte) and dextrorphan (IS) by erythromycin, as an example, which may cause over- or underestimation of CYP3A4 inhibition. To avoid this kind of effect, we proposed to use a stable isotope-labeled substrate and determine labeled metabolites by using unlabeled authentic compounds of each metabolite. We showed that CYP3A4 activity was determined with high accuracy and precision by using stable isotope-labeled midazolam even in the presence of an ion suppressor at high concentrations in the samples. This method is useful not only for the CYP inhibition screening but also for testing drug candidates to predict changes in metabolite formation by the possible co-administered drugs.
Nakamura
Koichi
K
Drug Metabolism & Physicochemistry Research Laboratory, R&D Division, Daiichi Pharmaceutical Co., Ltd.
Watanabe
Akiko
A
Okudaira
Noriko
N
Okazaki
Osamu
O
Sudo
Kenichi
K
D016477:Artifacts; D002247:Carbon Isotopes / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003917:Dextrorphan / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004917:Erythromycin / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D007654:Ketoconazole / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects* / Q000201:enzymology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D008874:Midazolam / Q000378:metabolism*; D015335:Molecular Probes / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000378:metabolism; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
D002247:Carbon Isotopes; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D015335:Molecular Probes; D009587:Nitrogen Isotopes; D003917:Dextrorphan; D004917:Erythromycin; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D008874:Midazolam; D007654:Ketoconazole