Assay for evaluation of proarrhythmic effects of herbal products: Case study with 12 Evodia preparations

Figures

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  Fig. 1. Effects of Evodia extracts on AP in hiPSC-CMs A, B Representative APs in control (blue lines) and after 5- or 30-minutes in the presence of EF6 (black lines) or EF7 (red lines). C, D Representative APs after 5- or 30-minutes incubation in EF5 (purple lines). E, Changes in APD90 (in %) after 5 min incubation with indicated extracts plotted versus respective DHE concentrations of the extract. F, Changes in APD90 (in %) after 30 min incubation with indicated extracts plotted versus respective DHE concentrations of the extract. See Supplementary Table 14S for the rate corrected action potential duration (APDc). Number of datapoints in A-F = 4. Dotted line in F marks the 50% threshold to make the division into the low- intermediate- and high-arrhythmogenic categories. Extracts in A – F were applied at a concentration of 10 mg/ml.

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  Fig. 2. Changes in AP triangulation in hiPSC-CMs plotted against DHE content of Evodia extracts Changes in triangulation after 5 min (A) or 30 min (B) incubation with indicated extracts plotted versus their DHE concentration (Fig. 2). Data points are coloured according to the assignment of the extracts to different groups in Table 1 (low-, intermediate- and high-arrhythmogenic). Estimated correlation coefficients R2 were 0.86 (A) and 0.88 (B). Extracts in A and B were applied at a concentration of 10 µg/ml. The most prominent triangulation was induced by EF5 and EP4 after 30 min exposure time (1736 ± 263, Table 7 S). However, oscillations during the plateau of the AP (Fig. 1 B) complicated the estimation of the APD90 prolongation and these data points were therefore not included in (B).

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  Fig. 3. hERG inhibition by Evodia extracts Concentration response curves of hERG inhibition by the high arrhythmogenic extract EF5 (IC50 = 8,7 ± 1,1 µg/ml, A) the intermediate arrhythmogenic extracts EF4 (IC50 = 12,1 ± 3,2 µg/ml, B) and EF7 (IC50 = 18,8 ± 3,6 µg/ml, C) and the low arrhythmogenic EP1 (IC50 = 83,1 ± 16,1 µg/ml, D).

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  Fig. 4. DHE and hortiamine prolong the FPD (A) Averaged traces on the left represent the independent, time matched, DMSO control field potential. The percent change in beat period (BP), field potential duration (FPD), and depolarisation spike amplitude (AMP) for five consecutive recordings is shown on the right. Control 1 combines the controls of the two MEAs treated with DHE, and control 2 – the control wells on the two chips treated with hortiamine. (B) The representative, averaged traces on the left visualise the shift of the t-wave and the prolongation of the FPD following treatment with DHE: blue (baseline), yellow (0.01 µM), black (0.03 µM), red (0.1 µM), and purple (0.3 µM). The relative changes in BP, FPD, and AMP, compared to baseline are shown on the right (n = 10). One-sample t-test was used, n.s. p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. (C) The representative traces on the left visualise the FPD following treatment with hortiamine. The percent changes to the three parameters (BP, FPD, AMP) are shown on the right (n = 10). One-sample t-test was used. See Supplementary Table 15 S for the beat rate corrected field potential duration (FPDc).

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  Fig. 5. Simulated AP of the human ventricular cardiomyocyte at steady state with pacing at 1 Hz according to the O’Hara-Rudy model [21].

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  Fig. 6. Torsadogenic probability of DHE and hortiamine calculated using the MICE model [13].

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  Fig. 7. Estimating proarrhythmic activity of herbal products Arrhythmogenic effects of plant extracts in hiPSC-CMs are detectable with MEA (e.g., by prolongations of the field potential or oscillations) or VSO technology (e.g., by prolongation of the APD90, triangulation or oscillations) (Fig. 1, Fig. 2, Fig. 4) [3]. Subsequent identification of hERG inhibitors e.g., by HPLC-based activity profiling; [23], estimation of their potency for hERG inhibition, and their content enables together with In-silico simulations (Fig. 5), a first judgement on potential proarrhythmic effects. Estimation of IC50 values for hERG, Cav1,.2, Nav1.5 and corresponding simulations of effects on the cardiac AP provide insights into the underlying mechanisms and the role of different ion channels. If the free plasma concentration of an identified arrhythmogenic compound (e.g., hERG inhibitor) is known, an extract can be considered arrhythmogenic, provided the IC50(hERG) and the FPC of identified hERG inhibitor(s) satisfy IC50(hERG) / FPC < 30 (see ICH S7B). If the free plasma concentration of hERG inhibitors is unknown, estimation of IC50 values for hERG, NaV1.2 and CaV1.2 inhibition enable the calculation of a…