Site-directed deuteration of dronedarone preserves cytochrome P4502J2 activity and mitigates its cardiac adverse effects in canine arrhythmic hearts

Figures

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  Figure 1. Deuteration of dronedarone preserves its metabolic and pharmacological activities but mitigates the inactivation of CYP2J2. Chemical structures of (A) amiodarone (left), dronedarone (middle) and poyendarone (right). Poyendarone is structurally identical to dronedarone except for the replacement of hydrogen atoms with deuterium, D on positions 4, 5,7 on benzofuran ring. (B) HLM metabolizes poyendarone (left) and dronedarone (right) to the same extent via the first-order kinetics indicated by linear decline in the logarithm of percentage drug remaining (y-axis) with increasing incubation time (x-axis). Similarly, (C) CYP2J2, (D) CYP3A4 and (E) CYP3A5 metabolizes poyendarone (left) and dronedarone (right) to the same extent via the first-order kinetics. (F) In vitro blockade of NaV1.5 (left), CaV1.2 (middle) and KV11.1 (right) currents by poyendarone (upper panel) or dronedarone (lower panel). The dose–response curve represents percentage blockade of each current against logarithm of concentration. The graphs were plotted using GraphPad Prism software v8.4.2 (San Diego, CA, USA).

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  Figure 2. Time-, concentration-dependent inactivation of CYP2J2, CYP3A4 and CYP3A5 activity by poyendarone and dronedarone with rivaroxaban as probe substrate. The logarithm of percentage (A) CYP2J2, (B) CYP3A4 and (C) CYP3A5 activity was plotted against pre-incubation time in the presence of poyendarone (left) and dronedarone (middle) with rivaroxaban as substrate. The observed inactivation rate (kobs) for CYP2J2, CYP3A4 or CYP2A5 was plotted against the inactivator concentration (right) to calculate kinact and KI for poyendarone and dronedarone (Table 2). Each point represents mean ± SEM of three experiments. The graphs were plotted using GraphPad Prism software v8.4.2 (San Diego, CA, USA).

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  Figure 3. Percentage of AA metabolized to each respective EET (y-axis) by CYP2J2 was plotted against increasing concentration of (A) poyendarone or (B) dronedarone (x-axis). Dronedarone inhibits CYP2J2-mediated AA metabolism in a concentration-dependent manner. Poyendarone prevents CYP2J2-mediated inhibition of AA metabolism and maintains EET levels at highest tested concentration. Each bar represents mean ± SEM of one experiment performed in triplicates. The different groups were compared with two-way ANOVA followed by Tukey's post-hoc correction and statistical significance was determined when ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, #P < 0.0001. Analysis was performed using GraphPad Prism software v8.4.2 (San Diego, CA, USA).

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  Figure 4. Poyendarone exhibits lower mitochondrial toxicity in isolated mitochondria. (A) Mitochondrial toxicity profiles in H9c2 cells incubated with increasing concentrations of poyendarone. Both cytotoxicity (black) and ATP levels (magenta) are expressed as percentages of control. Data represented as the mean ± SEM of experiments performed across three cell passages in triplicates. Increasing concentrations of poyendarone and dronedarone were compared with control (0 μmol/L) with one-way ANOVA followed by Dunnett's post-hoc correction. (B) Lack of inhibition of NADH- or succinate-supplemented ETC activity by poyendarone in isolated rat heart mitochondria. Rotenone (2 μmol/L) or malonic acid (1 mmol/L) was positive inhibitor for complex I and II, respectively. The graph represents the OCR (as % control), reflective of the activity of the mitochondrial complexes I and II inhibitor concentrations. Each bar shows mean ± SEM for three mitochondrial preparations. Different groups were compared with control with one-way ANOVA followed by Dunnett's post-hoc correction. Statistical significance was confirmed when ∗∗P < 0.01 two-way. Analysis was performed using GraphPad Prism software v8.4.2…

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  Figure 5. Dronedarone but not poyendarone inhibits CYP2J2 and causes BTB variability at physiological concentration. (A) The percentage of CYP2J2 gene expression in hiPSC-CMs relative to GAPDH. (B) Inhibition of CYP2J2 in hiPSC-CMs. Percentage CYP2J2 activity was plotted against 1 μmol/L poyendarone or dronedarone. Each bar represents mean ± SEM of three experiments performed in triplicates. The groups were compared with one-way ANOVA followed by Dunnett's post-hoc correction. (C) The Poincaré plots of control, 0.001–1 μmol/L poyendarone (upper panel) and dronedarone (lower panel). Points encompassed in an ellipse and aligned to line of identity indicate minimum BTB variability while scattered points indicate high BTB variability. (D) SD1 or STVRR and (E) SD2 or LTVRR of control, 0.001–1 μmol/L dronedarone and poyendarone. Each bar represents mean ± SEM of one experiment performed in triplicates. Different groups were compared with control with one-way ANOVA followed by Dunnett's post-hoc correction. Statistical significance was confirmed when ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, #P < 0.0001. Analysis was performed using GraphPad Prism software v8.4.2 (San Diego, CA, USA).

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  Figure 6. CYP2J2 knockdown (CYP2J2 KD) perturbed electrophysiological properties in cardiac cells. (A) qPCR was performed 96 h post-siRNA transfection where there was a 40%–80% knockdown of CYP2J2 compared to the control treatment across all siRNA treatments. Comparison were performed with one-way ANOVA followed by Dunnett's post hoc test (∗∗∗P < 0.001, n = 3). CYP2J2 KD exhibited higher (B) mean calcium transient duration (CTD), (C) mean beat-to-beat (BTB) interval and (D) BTB variability. (E) Poincaré plots were mapped where each point refers to the RR interval (beat cycle length) of nth+1 cardiac beat being plotted against nth beat. Poincaré plots: Control (left), CYP2J2 KD (right). Poincaré plot descriptors (F) short term variability (STVRR) and (G) long term variability (LTVRR) define the extent of variability. Data provided was expressed as mean ± SD. Each point represents the measured parameter of a single cardiomyocyte cluster measured within a 30 s window. Boxplots depict the median of the datasets, the lower and upper quartiles and the minimum or maximum values within 1.5 × interquartile range from the lower and upper quartiles, respectively. Number of clusters measured: Control,…

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  Figure 7. Poyendarone terminates persistent AF and exhibits lower torsadogenic risk. (A) Experimental plan for induction of atrioventricular block by catheter ablation and persistent AF by chronic atrial tachypacing at 600 bpm for more than 6 weeks in female beagle dogs. (B) Time courses of the anti-AF effects of poyendarone hydrochloride (n = 4), dronedarone hydrochloride (n = 4) and vehicle ethanol (n = 4). Antiarrhythmic effects of poyendarone (left) and dronedarone (right) on the conscious dogs with persistent AF. Typical tracings of Holter electrocardiogram (NASA lead: upper; and CM5 lead: lower in each panel). Poyendarone and dronedarone terminated the persistent AF at 6.1 and 5.2 h after their administration, respectively (arrows). Thereafter, poyendarone and dronedarone maintained the sinus rhythm in the dogs until 21 h after their administration. (C) Time courses of changes in QTcF, ΔQTcF, STVQT and ΔSTVQT after poyendarone and dronedarone administration. Time courses of the anti-AF effects, typical tracings of Holter electrocardiogram and time courses of changes in proarrhythmia parameters of amiodarone are depicted in Fig. S10. The QT interval was corrected with Fridericia's…