Mitochondrial toxicity of drugs

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

Mitochondrial Toxicity of Drugs

Mitochondria are important targets of drug toxicity. A variety of drugs has been shown to affect the electron transport chain, coupling of oxidative phosphorylation, ?-oxidation or other mitochondrial functions. Such damaging events may lead to the opening of a large pore across the mitochondrial membranes – the membrane permeability transition pore – eventually leading to apoptosis or necrosis of cells, depending on the cellular ATP content. Such drugs may therefore lead to organ damage, particularly in the liver, kidney, heart or skeletal muscle

Interaction with the hERG channel and cytotoxicity of amiodarone and amiodarone analogues

Amiodarone (2‐n‐butyl‐3‐[3,5 diiodo‐4‐diethylaminoethoxybenzoyl]‐benzofuran, B2‐O‐CH2CH2‐N‐diethyl) is an effective class III antiarrhythmic drug demonstrating potentially life‐threatening organ toxicity. The principal aim of the study was to find amiodarone analogues that retained human ether‐a‐go‐go‐related protein (hERG) channel inhibition but with reduced cytotoxicity. We synthesized amiodarone analogues with or without a positively ionizable nitrogen in the phenolic side chain. The cytotoxic properties of the compounds were evaluated using HepG2 (a hepatocyte cell line) and A549 cells (a pneumocyte line). Interactions of all compounds with the hERG channel were measured using pharmacological and in silico methods. Compared with amiodarone, which displayed only a weak cytotoxicity, the mono‐ and bis‐desethylated metabolites, the further degraded alcohol (B2‐O‐CH2‐CH2‐OH), the corresponding acid (B2‐O‐CH2‐COOH) and, finally, the newly synthesized B2‐O‐CH2‐CH2‐N‐pyrrolidine were equally or more toxic. Conversely, structural analogues such as the B2‐O‐CH2‐CH2‐N‐diisopropyl and the B2‐O‐CH2‐CH2‐N‐piperidine were significantly less toxic than amiodarone. Cytotoxicity was associated with a drop in the mitochondrial membrane potential, suggesting mitochondrial involvement. Pharmacological and in silico investigations concerning the interactions of these compounds with the hERG channel revealed that compounds carrying a basic nitrogen in the side chain display a much higher affinity than those lacking such a group. Specifically, B2‐O‐CH2‐CH2‐N‐piperidine and B2‐O‐CH2‐CH2‐N‐pyrrolidine revealed a higher affinity towards hERG channels than amiodarone. Amiodarone analogues with better hERG channel inhibition and cytotoxicity profiles than the parent compound have been identified, demonstrating that cytotoxicity and hERG channel interaction are mechanistically distinct and separable properties of the compounds

Hepatocellular toxicity and pharmacological effect of amiodarone and amiodarone derivatives

The aim of this work was to compare hepatocellular toxicity and pharmacological activity of amiodarone (2-n-butyl-3-[3,5 diiodo-4-diethylaminoethoxybenzoyl]-benzofuran; B2-O-Et-N-diethyl) and of eight amiodarone derivatives. Three amiodarone metabolites were studied, namely, mono-N-desethylamiodarone (B2-O-Et-NH-ethyl), di-N-desethylamiodarone (B2-O-Et-NH(2)), and (2-butyl-benzofuran-3-yl)-(4-hydroxy-3,5-diiodophenyl)-methanone (B2) carrying an ethanol side chain [(2-butylbenzofuran-3-yl)-[4-(2-hydroxyethoxy)-3,5-diiodophenyl]-methanone; B2-O-Et-OH]. In addition, five amiodarone analogs were investigated, namely, N-dimethylamiodarone (B2-O-Et-N-dimethyl), N-dipropylamiodarone (B2-O-Et-N-dipropyl), B2-O-carrying an acetate side chain [[4-(2-butyl-benzofuran-3-carbonyl)-2,6-diiodophenyl]-acetic acid; B2-O-acetate], B2-O-Et carrying an propionamide side chain (B2-O-Et-propionamide), and B2-O carrying an ethyl side chain [(2-butylbenzofuran-3-yl)-(4-ethoxy-3,5-diiodophenyl)-methanone; B2-O-Et]. A concentration-dependent increase in lactate dehydrogenase leakage from HepG2 cells and isolated rat hepatocytes was observed in the presence of amiodarone and of most analogs, confirming their hepatocellular toxicity. Using freshly isolated rat liver mitochondria, amiodarone and most analogs showed a dose-dependent toxicity on the respiratory chain and on beta-oxidation, significantly reducing the respiratory control ratio and oxidation of palmitate, respectively. The reactive oxygen species concentration in hepatocytes increased time-dependently, and apoptotic/necrotic cell populations were identified using flow cytometry and annexin V/propidium iodide staining. The effect of the three least toxic amiodarone analogs on the human ether-a-go-go-related gene (hERG) channel was compared with amiodarone. Amiodarone, B2-O-acetate, and B2-O-Et-N-dipropyl (each 10 microM) significantly reduced the hERG tail current amplitude, whereas 10 microM B2-O-Et displayed no detectable effect on hERG outward potassium currents. In conclusion, three amiodarone analogs (B2-O-Et-N-dipropyl, B2-O-acetate, and B2-O-Et) showed a lower hepatocellular toxicity profile than amiodarone, and two of these analogs (B2-O-Et-N-dipropyl and B2-O-acetate) retained hERG channel interaction capacity, suggesting that amiodarone analogs with class III antiarrhythmic activity and lower hepatic toxicity could be developed