Drug interactions and hepatotoxicity of clopidogrel

Clopidogrel (Plavix¨) is an antiplatelet drug, which is clinically used in combination with aspirin to reduce cardiovascular events in patients undergoing percutaneous coronary intervention or in patients suffering from acute coronary syndromes. Clopidogrel is a prodrug requiring enzymatic activation by cytochrome P450 (CYP) isoenzymes in order to inhibit platelet aggregation. Drug-interactions can affect clopidogrel activation and therefore cause interference with its pharmacodynamic effect. Clopidogrel is generally well tolerated but hepatotoxicity associated with clopidogrel treatment has been reported.
In the first project we investigated potential drug interactions of CYP inhibitors or substrates with the activation of clopidogrel or the inhibition of clopidogrelÕs antiplatelet effect. Using human liver microsomes (HLM) or specific supersomes we reproduced activation of clopidogrel in vitro. We found that CYP3A4 is primarily responsible for the metabolism of clopidogrel. At low concentrations (< 10 µM) CYP2C19 may contribute to clopidogrel activation to a certain degree. Additionally, HLM in co-incubation with platelets freshly prepared from human blood, allowed us to investigate the pharmacodynamic effect of clopidogrel ex vivo. We showed that activated clopidogrel dose-dependently inhibited platelet aggregation, whereas platelet aggregation occurred in absence of HLM. Potent CYP3A4 inhibitors exhibited strong inhibitory effects on clopidogrel activation and on clopidogrelÕs antiplatelet effect. Additionally, statins metabolized by CYP3A4 impaired clopidogrel activation and its antiplatelet effect. Since clopidogrel itself inhibited CYP2C19 at concentrations < 10µM, the CYP2C19 inhibitor lansoprazole affected clopidogrel biotransformation only at clopidogrel concentrations ²10 µM.
In the second project, we investigated whether the reactive metabolite of clopidogrel is responsible for the observed hepatotoxicity and studied the corresponding mechanism. Our liver toxicity models involved HepG2 cells that overexpress human CYP3A4 or were supplemented with CYP3A4 supersomes. These cellular systems were able to generate the active metabolite of clopidogrel, which was associated with cytotoxicity. Co-incubation with ketoconazole, a CYP3A4 inhibitor, attenuated the toxic effect, thereby confirming the CYP3A4 dependency of clopidogrel activation. Cytotoxicity was associated with the induction of an oxidative stress reaction and promoted apoptosis via a mitochondrial pathway. In contrast, the carboxylate metabolite of clopidogrel, which is generated by esterases after oral administration, did not cause cytotoxicity.
Amiodarone (Cordarone¨) is a class III antiarrhythmic drug used for the treatment of a wide spectrum of cardiac arrhythmias. AmiodaroneÕs therapeutic use is limited due to its numerous side effects such as liver toxicity. Recent in vitro investigations revealed that the N-desethyl metabolites of amiodarone may be partially responsible for the hepatotoxicity. Since CYP3A4 is responsible for amiodarone metabolism, CYP3A4 induction may represent an important risk factor in the clinic.
The aim of the third project was to study the role of CYP3A4 in amiodarone-induced hepatotoxicity. Experiments were conducted using the same cellular activation systems as for clopidogrel, since CYP3A4 is also responsible for amiodarone metabolization in the liver. The systems proved to be powerful screening tools for CYP3A4-mediated toxicity of xenobiotics. We demonstrated that amiodarone metabolization is CYP3A4 dependent and generates MDEA and DDEA. Accordingly, MDEA and DDEA are primarily responsible for the observed cytotoxicity by increasing ROS production, by inducing mitochondrial damage and cytochrome c release and by promoting late apoptosis/necrosis. Therefore, we concluded that induced activity of CYP3A4 is a risk factor for hepatotoxicity associated with amiodarone.