Mitochondrial toxicity of drugs

Abstract

The introduction of this thesis concentrates on the cellular energy supplier and an important instrument in mediating cell death, mitochondrion. First, an overview is given to explain the biochemical properties of this organelle. Then, the role of mitochondria in cell death is discussed, followed by an article about the mitochondrial toxicity of drugs. As an example of the mitochondrial toxicity of a drug, an article about statins and their effects on L6 myocytes and rat muscle mitochondria conclude the introduction part of this thesis. The aim of the first project was to compare hepatocellular toxicity and pharmacological activity of amiodarone (B2-O-Et-N-diethyl) and eight amiodarone derivatives, including three amiodarone metabolites (B2-O-Et-NH-ethyl, B2-O-Et-NH2 and B2-O-Et-OH). In addition, five amiodarone analogues were investigated (B2-O-Et-N-dimethyl, B2-O-Et-N-dipropyl, B2-OAcetate, B2-O-Et-propionamide and B2-O-Et). The studies were accomplished using frehly isolated rat liver mitochondria, primary rat hepatocytes and the hepatoma cell line HepG2. The hepatocellular toxicity of amiodarone and most of the derivatives was confirmed. Amiodarone and most analogues showed a dose-dependent toxicity on the respiratory chain and on β- oxidation of the mitochondria. The ROS concentration in hepatocytes increased timedependently and apoptotic/necrotic cell populations were identified using flow cytometry and annexinV/propidiumiodide staining. The effect of the three least toxic amiodarone analogues on the hERG channel was compared to amiodarone. In conclusion, three amiodarone analogues (B2-O-Et-N-dipropyl, B2-O-Acetate and B2-O-Et) showed a lower hepatocellular toxicity profile than amiodarone and two of these analogues (B2-O-Et-N-dipropyl and B2-O-Acetate) retained hERG channel interaction capacity, suggesting that amiodarone analogues with class III antiarrhythmic activity and lower hepatic toxicity could be developed. For the second project in this thesis, we synthesized three more amiodarone analogues (B2-O-Ethylacetate, B2-O-Et-N-pyrrolidine and B2-O-Et-N-piperidine) and, together with amiodarone and its metabolites (B2-O-Et-NH-ethyl and B2-O-Et-NH2) and some derivatives from the first study (B2-O-Et-N-dipropyl, B2-O-Et-propionamide, B2-O-Acetate, B2-O-Et-OH), characterized their hepatic toxicity together with the pulmonary toxicity. The interaction with the hERG channel was determined for all the derivatives. Compared to amiodarone, which showed only a weak cytotoxicity, the desethylated metabolites, B2-O-Acetate, B2-O-Et-OH and B2-O-Et- N-pyrrolidine showed a similar or higher cytotoxicity. On the other hand, B2-O-Et-N-dipropyl, B2-O-Ethylacetate, B2-O-Et, B2-O-Et-propionamide and B2-O-Et-N-piperidine were less toxic. Cytotoxicity was associated with a drop in the mitochondrial membrane potential and therefore most probably mitochondrial in origin. Substances carrying a nitrogen in the side chain (amiodarone, B2-O-Et-NH-ethyl, B2-O-Et-NH2, B2-O-Et-N-dipropyl, B2-O-Et-propionamide, B2-O-Et-N-pyrrolidine, B2-O-Et-N-piperidine) showed a much higher affinity to the hERG channel (range 0.22-12.2µmol/L) than those without a nitrogen in this position (B2-O-Acetate, B2-OEthylacetate, B2-O-Et-OH) (range 74-216µmol/L). Neither cytotoxicity, nor the interaction with the hERG channel, was associated with the lipophilicity of the compounds. It was concluded, that the physicochemical properties of amiodarone and its analogues were not as important for the potassium channel interaction and cytotoxicity as the chemical structure of the compounds. In the third project of this thesis, the relationship between an unexpected toxicity of a drug and an underlying mitochondrial defect was studied using human dermal fibroblasts. These cells were derived from patients suffering from a mitochondrial defect. Simvastatin and benzbromarone are known to cause an unexpected adverse reaction (myotoxicity or hepatotoxicity, respectively). Both dermal fibroblasts with a mitochondrial defect and fibroblasts from healthy patients were treated with different concentrations of benzbromarone and simvastatin, and the overall toxicity was evaluated after different time points. There were no differences in the toxicity pattern between the cell lines, and the toxicity assayed was relatively scarce in all experiments. It was concluded that the the test system was not suitable for these studies and that they should be repeated with other cell lines of hepatic or muscle origin.