Effect of Ethanol on S-Warfarin and Diclofenac Metabolism by Recombinant Human CYP2C9.1

Abstract

Warfarin, which consists of a racemic mixture of S-and Renantiomer, has been used as an anticoagulant agent. The anticoagulant activity of S-warfarin is 3-5 times greater than that of R-warfarin.1) This drug has a narrow therapeutic index and shows marked drug-drug interactions when coadministered with other agents that alter warfarin metabolism.2,3) The stereoselective metabolism of warfarin enantiomers in humans has been shown to be catalyzed by cytochrome P450 (CYP) such as CYP1A1/2, 2C9, and 3A4. 4,5) The 7-hydroxylation of S-warfarin is exclusively catalyzed by CYP2C9. RWarfarin is metabolized primarily by CYP1A2 to 6-and 8-hydroxywarfarin, and by CYP3A4 to 10-hydroxywarfarin. Diclofenac, a non-steroidal anti-inflammatory drug (NSAID), is also metabolized to 4Ј-hydroxydiclofenac by CYP2C9. 6,7)Ethanol is widely used as a pharmaceutical excipient for the solubilization of many hydrophobic drugs. Various drugs can pharmacokinetically or pharmacodynamically interact with alcohol. 8) Our previous study suggested that ethanol changes the binding of warfarin enantiomer to human serum albumin stereoselectively. 9) Acute intoxication by alcohol reduces the metabolism of warfarin by CYPs, causing increased anticoagulant effects, and leading to a risk of hemorrhage.10) Conversely, chronic alcohol ingestion might enhance metabolic enzyme activity, leading to a decrease in the anticoagulant effects of warfarin. Hamitouche et al. 11) showed that CYP2C9, as well as other CYPs such as CYP2E1, CYP1A2, and CYP3A4, is also able to metabolize ethanol, and indicated that these CYPs have a low affinity for ethanol for its metabolization. The apparent K m value determined for ethanol oxidation by various CYPs was around 10 mM. 11)Therefore, it is possible that ethanol affects the metabolism of other substrates by CYP2C9. Few studies on the effect of ethanol on warfarin metabolism by human CYP2C9 in vitro have been reported. The non-significant metabolic interaction of diclofenac (15 mM) with ethanol by CYP2C9 has been reported. 12) However, the effect of ethanol on the metabolism of diclofenac at lower concentrations by CYP2C9 has not been examined. In this paper, we have examined the effect of ethanol on the metabolism of S-warfarin and diclofenac by recombinant CYP2C9.1 microsomes (CYP2C9.1). MATERIALS AND METHODS Materials S-(Ϫ)-Warfarin, diclofenac sodium, ethanol, naproxen, NADPϩ , glucose-6-phosphate, MgCl 2 · 6H 2 O, and glucose-6-phosphate dehydrogenase were purchased from Wako Pure Chemical Ind. (Osaka, Japan). 4Ј-Hydroxydiclofenac was purchased from Sigma-Aldrich (MO, U.S.A.). 7-Hydroxywarfarin and human CYP2C9.1ϩP450 reductase microsomes derived from baculovirus expression systems were purchased from BD Gentest (MA, U.S.A.). All other chemicals and solvents were of analytical grade or higher.Inhibition Study According to the method of Iwakawa et al., 13) a metabolic inhibition study was performed. The time of incubation and concentration of microsomal protein in the study were determined to be in a linear range for ...

Influence ofCYP2C9andVKORC1Polymorphisms on Warfarin and Acenocoumarol in a Sample of Lebanese People

“…In our population, acenocoumarol final weekly maintenance dose was affected by age, target INR range, and alcohol intake. All research participants who reported alcohol use were occasional drinkers, and none had evidence of liver disease; note that it has been previously shown that ethanol may inhibit drug metabolism by CYP2C9 41 , 42 . The use of the CYP2C9 inhibitor amiodarone did not play a major role; it is possible that this is due to the low number of patients taking this drug.…”

Substrates, Inducers, Inhibitors and Structure-Activity Relationships of Human Cytochrome P450 2C9 and Implications in Drug Development

“…However, the inhibitory effect of ethanol on the 4'-hydroxylation of diclofenac was not observed even at 1.0 vol% (170 mM) ethanol. Ethanol at a concentration of 3.0 vol% (510 mM) increased the K m value of diclofenac metabolism without changes in the V max , which indicates that diclofenac 4'-hydroxylation by CYP2C9 was competitively inhibited by ethanol [1367]. These results indicate that S-warfarin metabolism by CYP2C9 is more sensitive to ethanol than diclofenac metabolism and ethanol inhibits the metabolism by CYP2C9 in a substratedependent manner.…”

“…In recombinant CYP2C9, the 7-hydroxylation of S-warfarin was inhibited by as low as 0.1 vol% (17 mM) ethanol [1367]. Ethanol decreased the V max /K m and V max values of S-warfarin metabolism in a concentration-dependent manner, but the K m value was unchanged by ethanol.…”

Noncompetitive inhibition of human CYP2C9 in vitro by a commercial Rhodiola rosea product

“…In fact, the influence of ethanol on CYP2C9 activity seems to be substrate dependent, with a significant inhibition on warfarin metabolism at 0.1 vol% and no inhibition of diclofenac metabolism at 1 vol% (Tatsumi et al. ). In general, the influence of ethanol on CYP2C9 differs from that on CYP2D6, where Hellum and Nilsen () found a biphasic effect with a small inhibition at 0.1%, a significant activation at 0.5%, 0.8%, 1.1%, 1.5%, and 5%, and a significant inhibition at 8% and 15%.…”