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

Abstract

Background and purpose: Amiodarone (2-n-butyl-3-[3,5 diiodo-4-diethylaminoethoxybenzoyl]-benzofuran, B2-O-CH(2)CH(2)-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.

Experimental approach: 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.

Key results: Compared with amiodarone, which displayed only a weak cytotoxicity, the mono- and bis-desethylated metabolites, the further degraded alcohol (B2-O-CH(2)-CH(2)-OH), the corresponding acid (B2-O-CH(2)-COOH) and, finally, the newly synthesized B2-O-CH(2)-CH(2)-N-pyrrolidine were equally or more toxic. Conversely, structural analogues such as the B2-O-CH(2)-CH(2)-N-diisopropyl and the B2-O-CH(2)-CH(2)-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-CH(2)-CH(2)-N-piperidine and B2-O-CH(2)-CH(2)-N-pyrrolidine revealed a higher affinity towards hERG channels than amiodarone.

Conclusions and implications: 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.

Figure 1

Adenylate kinase release from HepG2 cells treated with amiodarone or amiodarone analogues. B2-O-CH₂CH₂-NH-ethyl, B2-O-CH₂CH₂-NH₂, B2-O-CH₂-COOH and B2-O-CH₂CH₂-N-pyrrolidine showed cytotoxicity starting at 10 μM, amiodarone and B2-O-CH₂CH₂-N-piperidine starting at 100 μM, and B2-O-CH₂CH₂-N-diisopropyl and B2-O-CH₂-COO-CH₂CH₃ no cytotoxicity up to 100 μM. Simvastatin was used as a positive control. All samples contained 0.1% dimethyl sulphoxide (DMSO) (except medium only). Data are presented as means±s.e.mean of at least four incubations in triplicate. ^*P<0.05 and ^**P<0.01 versus control incubations containing 0.1% DMSO.

Figure 2

Inhibition of reductive capacity of HepG2 cells (resazurin reduction test) by amiodarone or amiodarone derivatives. B2-O-CH₂CH₂-NH₂ inhibited reductive capacity starting at 10 μM, B2-O-CH₂CH₂-NH-ethyl, B2-O-CH₂-COOH and B2-O-CH₂CH₂-N-pyrrolidine at 100 μM, and amiodarone, B2-O-CH₂CH₂-N-diisopropyl, B2-O-CH₂-COO-CH₂CH₃ and B2-O-CH₂CH₂-N-piperidine revealed no inhibition up to 100 μM. Simvastatin was used as a positive control. All samples contained 0.1% dimethyl sulphoxide (DMSO) (except medium only). Data are presented as means±s.e.mean of at least four incubations in triplicate. ^*P<0.05 and ^**P<0.01 versus control incubations containing 0.1% DMSO.

Figure 3

Adenylate kinase release from A549 cells treated with amiodarone or amiodarone derivatives. B2-O-CH₂CH₂-NH-ethyl and B2-O-CH₂CH₂-NH₂ showed cytotoxicity starting at 10 μM, amiodarone, B2-O-CH₂-COOH and B2-O-CH₂CH₂-N-pyrrolidine at 100 μM, and B2-O-CH₂CH₂-N-diisopropyl, B2-O-CH₂-COO-CH₂CH₃ and B2-O-CH₂CH₂-N-piperidine no cytotoxicity up to 100 μM. Simvastatin was used as a positive control. All samples contained 0.1% dimethyl sulphoxide (DMSO) (except medium only). Data are presented as means±s.e.mean of at least four incubations in triplicate. ^**P<0.01 versus control incubations containing 0.1% DMSO.

Figure 4

Inhibition of reductive capacity of A549 cells (resazurin reduction test) by amiodarone or amiodarone derivatives. B2-O-CH₂CH₂-NH-ethyl inhibited reductive capacity starting at 10 μM, B2-O-CH₂CH₂-NH₂ and B2-O-CH₂-COOH at 100 μM, and amiodarone, B2-O-CH₂CH₂-N-diisopropyl, B2-O-CH₂-COO-CH₂CH₃, B2-O-CH₂CH₂-N-pyrrolidine and B2-O-CH₂CH₂-N-piperidine revealed no inhibition up to 100 μM. Simvastatin was used as a positive control. All samples contained 0.1% dimethyl sulphoxide (DMSO) (except medium only). Data are presented as means±s.e.mean of at least four incubations in triplicate. ^*P<0.05 and ^**P<0.01 versus control incubations containing 0.1% DMSO.

Figure 5

Dose–response curves of amiodarone and amiodarone derivatives. Amiodarone, the amiodarone metabolites B2-O-CH₂CH₂-NH-ethyl and B2-O-CH₂CH₂-NH₂, and the analogues B2-O-CH₂CH₂-N-pyrrolidine and B2-O-CH₂CH₂-N-piperidine revealed strong hERG inhibition with IC₅₀<1 μM. B2-O-CH₂CH₂-N-diisopropyl and B2-O-CH₂CH₂-NH-CO-CH₂CH₃ were medium strong inhibitors (IC₅₀>1 and <10 μM), whereas B2-O-CH₂-COOH, B2-O-CH₂CH₂-OH and B2-O-CH₂-COO-CH₂CH₃ showed only a weak inhibition. For B2-O-CH₂CH₂-OH, the IC₅₀ was not calculated as the inhibition of the hERG channel was <50% at 100 μM. For B2-O-CH₂CH₃ (not shown in the figure), no inhibition of the hERG channel was detectable up to 10 μM and higher concentrations could not be tested due to solubility problems. Measurements were accomplished in the whole-cell patch-clamp configuration at room temperature. Outward currents were activated upon depolarization of the cell membrane from −80 to +20 mV for 3 s, whereas partial repolarization to −40 mV for 4 s evoked large tail currents. At least three cells were recorded per test compound.

Figure 6

Inhibition of the potassium current by B2-O-CH₂CH₂-N-diethyl (amiodarone (1)), B2-O-CH₂-COO-CH₂CH₃ (11), B2-O-CH₂CH₂-N-diisopropyl (4) and B2-O-CH₂CH₂-N-piperidine (9). Representative traces of potassium currents across hERG channels stably expressed in HEK Tet cells are shown. Measurements were accomplished in the whole-cell patch-clamp configuration at room temperature. Outward currents were activated upon depolarization of the cell membrane from −80 to +20 mV for 3 s, whereas partial repolarization to −40 mV for 4 s evoked large tail currents. At least three cells were recorded per test compound. The vehicle (0.1% dimethyl sulphoxide (DMSO)) had no significant effect on hERG channel activity. In contrast, 10 μM B2-O-Et-N-diethyl (amiodarone (1)) as well as 10 μM B2-O-CH₂CH₂-N-piperidine (9) blocked the hERG channel completely. In comparison, the interaction of 10 μM B2-O-CH₂-COO-CH₂CH₃ (11) as well as 10 μM B2-O-CH₂CH₂-N-diisopropyl (4) with the hERG channel was less intense. The upper trace in the individual figures depicts the control incubations (0.1% DMSO) and the lower trace the incubations containing the test compounds in 0.1% DMSO.

Figure 7

(a) Amiodarone (yellow) is docked into the vestibule of a hERG model. Two of the four chains of the hERG homo-tetramer are shown in C-α representation (green, magenta). The Connolly surface was produced with a probe radius of 1.4 Å. The selectivity filter cannot be displayed in this representation due to its very narrow diameter. A white ball represents a potassium ion in the filter region as found in the KscA structure template (Morais-Cabral et al., 2001). (b) Amiodarone derivative 5 (blue) is docked in a hypothetical position similar to the one assumed by 1. From the apparent positional similarity of 1 and 5, it can be deduced that the positively charged nitrogen (seen in 1 as the blue ball) has an attractive effect. The negatively charged carboxylate of 5, being in a position similar to that of the positively charged nitrogen in 1, induces strong repulsive effects.