Strategies for Determining Correct Cytochrome P450 Contributions in Hepatic Clearance Predictions: In Vitro-In Vivo Extrapolation as Modelling Approach and Tramadol as Proof-of Concept Compound.
OBJECTIVE
Although the measurement of cytochrome P450 (CYP) contributions in metabolism assays is straightforward, determination of actual in vivo contributions might be challenging. How representative are in vitro for in vivo CYP contributions? This article proposes an improved strategy for the determination of in vivo CYP enzyme-specific metabolic contributions, based on in vitro data, using an in vitro-in vivo extrapolation (IVIVE) approach. Approaches are exemplified using tramadol as model compound, and CYP2D6 and CYP3A4 as involved enzymes.


METHODS
Metabolism data for tramadol and for the probe substrates midazolam (CYP3A4) and dextromethorphan (CYP2D6) were gathered in human liver microsomes (HLM) and recombinant human enzyme systems (rhCYP). From these probe substrates, an activity-adjustment factor (AAF) was calculated per CYP enzyme, for the determination of correct hepatic clearance contributions. As a reference, tramadol CYP contributions were scaled-back from in vivo data (retrograde approach) and were compared with the ones derived in vitro. In this view, the AAF is an enzyme-specific factor, calculated from reference probe activity measurements in vitro and in vivo, that allows appropriate scaling of a test drug's in vitro activity to the 'healthy volunteer' population level. Calculation of an AAF, thus accounts for any 'experimental' or 'batch-specific' activity difference between in vitro HLM and in vivo derived activity.


RESULTS
In this specific HLM batch, for CYP3A4 and CYP2D6, an AAF of 0.91 and 1.97 was calculated, respectively. This implies that, in this batch, the in vitro CYP3A4 activity is 1.10-fold higher and the CYP2D6 activity 1.97-fold lower, compared to in vivo derived CYP activities.


CONCLUSIONS
This study shows that, in cases where the HLM pool does not represent the typical mean population CYP activities, AAF correction of in vitro metabolism data, optimizes CYP contributions in the prediction of hepatic clearance. Therefore, in vitro parameters for any test compound, obtained in a particular batch, should be corrected with the AAF for the respective enzymes. In the current study, especially the CYP2D6 contribution was found, to better reflect the average in vivo situation. It is recommended that this novel approach is further evaluated using a broader range of compounds.
Laboratory of Medical Biochemistry and Clinical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000, Ghent, Belgium. huybrecht.tjollyn@ugent.be.
Huybrecht
http://orcid.org/0000-0003-2101-9926
H
T'jollyn
Janssen Research and Development, A Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340, Beerse, Belgium.
Jan
J
Snoeys
Laboratory of Medical Biochemistry and Clinical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000, Ghent, Belgium.
Jan
J
Van Bocxlaer
Laboratory of Medical Biochemistry and Clinical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000, Ghent, Belgium.
Lies
L
De Bock
Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O&N2, Herestraat 49, Box 921, 3000, Louvain, Belgium.
Pieter
P
Annaert
Janssen Research and Development, A Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340, Beerse, Belgium.
Achiel
A
Van Peer
Department of Development and Regeneration, KU Leuven and Neonatal Intensive Care Unit, University Hospital Leuven, 3000, Louvain, Belgium.
Karel
K
Allegaert
Janssen Research and Development, A Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340, Beerse, Belgium.
Geert
G
Mannens
Laboratory of Medical Biochemistry and Clinical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000, Ghent, Belgium.|Janssen Research and Development, A Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340, Beerse, Belgium.
An
A
Vermeulen
Laboratory of Medical Biochemistry and Clinical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000, Ghent, Belgium.
Koen
K
Boussery
D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D003915:Dextromethorphan / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008657:Metabolic Clearance Rate / Q000502:physiology*; D008862:Microsomes, Liver / Q000378:metabolism; D008874:Midazolam / Q000378:metabolism; D014147:Tramadol / Q000378:metabolism*
D014147:Tramadol; D003915:Dextromethorphan; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D008874:Midazolam