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. 2016 Aug 1;13(8):2833-43.
doi: 10.1021/acs.molpharmaceut.6b00436. Epub 2016 Jul 1.

Mechanism-Based Inhibition of CYP3A4 by Podophyllotoxin: Aging of an Intermediate Is Important for in Vitro/in Vivo Correlations

Carlo Barnaba  1 , Jaydeep Yadav  2 , Swati Nagar  2 , Ken Korzekwa  2 , Jeffrey P Jones  1
Affiliations

Mechanism-Based Inhibition of CYP3A4 by Podophyllotoxin: Aging of an Intermediate Is Important for in Vitro/in Vivo Correlations

Carlo Barnaba et al. Mol Pharm. .

Abstract

An in vitro observation of time-dependent inhibition (TDI) of metabolic enzymes often results in removing a potential drug from the drug pipeline. However, the accepted method for predicting TDIs of the important drug metabolizing cytochrome P450 enzymes often overestimates the drug interaction potential. Better models that take into account the complexities of the cytochrome P450 enzyme system will lead to better predictions. Herein we report the use of our previously described models for complex kinetics of podophyllotoxin. Spectral characterization of the kinetics indicates that an intermediate MI complex is formed, which slowly progresses to an essentially irreversible MI complex. The intermediate MI complex can release free enzyme during the time course of a typical 30 min TDI experiment. This slow rate of MI complex conversion results in an overprediction of the kinact value if this process is not included in the analysis of the activity versus time profile. In vitro kinetic experiments in rat liver microsomes predicted a lack of drug interaction between podophyllotoxin and midazolam. In vivo rat pharmacokinetic studies confirmed this lack of drug interaction.

Keywords: carbene; mechanism-based inhibition; methylenedioxyphenyl compounds; podophyllotoxin; time-dependent inhibition.

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Figures

Figure 1
Figure 1
Mechanism-based inhibition of cytochrome P450 by methylenedioxyphenyl compounds.
Figure 2
Figure 2
Spectra of MI- complex formation upon metabolism of PPT by CYP3A4. (a) Raw spectra for metabolite complex formation in CYP3A4 baculosomes. (b) Principal spectral components (we attribute PC1 to the Fe2+:carbene complex, PC2 to the oxidized intermediate Fe3+:carbene and PC3 to cytochrome P420) and (c) dynamic components obtained through SVD analysis of raw spectra. (d) Raw spectra for metabolite complex formation in HLM. (e) Principal spectral components and (f) dynamic components obtained through SVD analysis of raw spectra.
Figure 3
Figure 3
Proposed kinetic scheme for MIC formation. The species depicted in the schemes are defined as follows: E, unbound active enzyme; S, substrate; ES, enzyme-susbtrate complex; Fe3+:carbene, ferricytochrome:carbene complex (absorbance maximum at 437 nm); Fe2+:carbene, ferrocytochrome:carbene complex (absorbance maximum at 455 nm); P420, cytochrome P420 (absorbance maximum at 425 nm); Met, PPT metabolite derived from carbene release.
Figure 4
Figure 4
Numerical fitting of the spectral complexes for CYP3A4-mediated metabolism of PPT in (a) baculosomes and (b) HLM.
Figure 5
Figure 5
Numerical fitting of the TDI in vitro data (HLM). ESI-QI scheme (a), standard replot for MM kinetics (b), ESI-Q concentration-time plot (c), ESI-Q PRA plot (d); ESI-QI concentration-time plot (e) ESI-QI PRA plot (f). E* = Fe3+:carbene; E**= Fe2+:carbene; enzyme loss (k’9) occurs from E, ES, EI, and ESI.
Figure 6
Figure 6
Numerical fitting of the TDI in vitro data (RLM). MM-Q scheme (a), MM-Q concentration-time plot (b), MM-Q PRA plot (c); EII-Q scheme (d), EII-Q concentration time plot (e) and EII-Q PRA plot (f). The QI scheme is depicted but reduces to the Q-model since k5 parameterized to zero.
Figure 7
Figure 7
Concentration-time profile for MDZ in rats, with and without PPT.
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