Warfarin is an anticoagulant used for the prevention of thromboembolic events. It has a narrow therapeutic index and, therefore, compounds affecting its metabolism could have serious consequences. Available data indicate that warfarin is mostly metabolized by CYP-2C9, with the formation of hydroxylated metabolites. Amiodarone is a widely used antiarrhythmic drug which inhibit different CYP, including CYP2C9.
Here, we exploited Control SilensomesTM and CYP-2C9 SilensomesTM to study the involvement of CYP-2C9 in the metabolism of S-warfarin, and to study the effects of amiodarone on this metabolism.
MATERIALS AND METHODS
SilensomesTM are provided by Biopredic International, France.
S-warfarin (WRF) (1 uM) was incubated, in absence or presence of 5 μM amiodarone, with Control SilensomesTM and CYP2C9 SilensomesTM, for 30 min at 37°C. The different WRF metabolites, and in particular the hydroxylated ones, were identified and quantified by an analytical method based on liquid chromatography - triple quadrupole mass spectrometry (HPLC-MS/MS).
The data obtained with control SilensomesTM confirmed that hydroxylation is the main metabolic pathway of S-WRF with 7-OH-WRF being the most abundant metabolite followed by 6-OH-WRF and 4-OH-WRF; other peaks observed in the chromatograms were negligible.
When S-WRF was incubated with CYP-2C9 SilensomesTM, the levels of 7-OH-WRF were 73±3% lower; since 2C9- SilensomesTM have 82% inhibition of 2C9 (Biopredic, certificate of analysis), it follows that CYP-2C9 mediates about 90% of 7-OH-formation, in agreement with the literature (Rettie et al., 1992). The levels of 6-OH-WRF were 40±3% lower with CYP-2C9 SilensomesTM, indicating that only ~50% of 6-OH-WRF formation is mediated by CYP-2C9. The data indicate that this CYP is not involved in the (very low) formation of 4-OH-WRF.
Amiodarone significantly inhibited the CYP-2C9-mediated formation of 7-OH-WRF: as expected, a very similar inhibitory effect was observed on total CYP activity (control SilensomesTM) and on specific CYP-2C9 activity (Control SilensomesTM minus CYP-2C9 SilensomesTM ).
On the contrary, the formation of 6-OH-WRF is only partly (50%) due to CYP-2C9, and the remaining possibly due to CYP-1A2 and CYP-3A4 (Rettie et al., 1992). The data obtained that is evaluating the effect of amiodarone in Control Silensomes TM and CYP-2C9 Silensomes TM , clearly indicate that this drug inhibits 6-OH-formation mainly acting on a CYP other than CYP-2C9. This is an original observation, made possible by the use of SilensomesTM.