Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells

Figure 1. hERG K+ current IC50 concentrations. Graphical representation of hERG K+ current inhibition by the 12 test compounds: spread of individual log-IC50 concentrations per compound and per site (compare Supplementary Tables S2–S4) with superimposed box-and-whiskers plot (median, 25/75 percentile, min/max values); the number of data points from automated platforms is indicated at the bottom of each column. Platforms used are color-coded as follows: p1, blue; p2, green; p3, orange; p4, black; and p5, black. Assays (s01–s19) were conducted at room temperature (open symbols) and at 35 °C (filled symbols). bep, bepridil; cpz, chlorpromazine; cis, cisapride; dil, diltiazem; dof, dofetilide; mex, mexiletine; ond, ondansetron; qui, quinidine; ran, ranolazine; sot, sotalol; ter, terfenadine; ver, verapamil.

Figure 2. Variability of hERG K+ current inhibition at NOEL/LOEL concentrations per site. Test compound concentrations were chosen in such a manner that the lowest concentration for each compound was known/expected to cause no (non-inhibitory, no observable effect level [NOEL]) or only a minor inhibition (lowest observable effect level [LOEL]) of the hERG K+ current. Therefore, at these NOEL/LOEL concentrations, current inhibition values are expected to cluster around zero percent. Inhibition mean values (and SD) at the lowest test compound concentration were averaged per site over all 12 test compounds. Platforms used are color-coded as follows: p1, blue; p2, green; p3, orange; p4, black; and p5, black. Automated assays (s01–s19) were conducted at room temperature (open symbols) and at 35 °C (filled symbols).

Figure 3. Peak hNav1.5 Na+ current IC50 concentrations per compound. Graphical representation of peak hNav1.5 Na+ current inhibition by the 12 test compounds: spread of individual log-IC50 concentrations per compound and per site (compare Supplementary Tables S5–S7) with superimposed box-and-whiskers plot (median, 25/75 percentile, min/max values); the number of data points from automated platforms is indicated at the bottom of each column. Platforms used are color-coded as follows: p1, blue; p2, green; p3, orange; p4, black; and p5, black. Automated assays (s01–s19) were conducted at room temperature (open symbols) and at 35 °C (filled symbols). No inhibition defined as no obvious concentration-dependent current block and/or no curve fitting performed. bep, bepridil; cpz, chlorpromazine; cis, cisapride; dil, diltiazem; dof, dofetilide; mex, mexiletine; ond, ondansetron; qui, quinidine; ran, ranolazine; sot, sotalol; ter, terfenadine; ver, verapamil.

Figure 4. Platform variability for hERG and peak Nav1.5 current block. A comparison of platform variability for dofetilide block of hERG (a) and mexiletine block of hNav1.5 current (b). Despite limited data, minimal variability is evident for block of both currents with platform 3, while prominent differences in variability for block of hERG vs. peak hNav1.5 current for platform 2 are observed. Note the logarithmic scale used to illustrate the wide range of values obtained within and between platforms.

Figure 5. A comparison of between- and within-platform variance and repeatability coefficients for hERG and hNav1.5 currents. (a) For hERG, within-platform variance predominated for 6 of 12 drugs (cisapride, mexiletine, ondansetron, quinidine, sotalol, and verapamil). (c) For hNav1.5, within-platform variance predominated for 2 of 6 drugs (diltiazem and mexiletine). (b,d) Repeatability coefficients for hERG and hNav1.5 current block differed widely across automated patch platforms from which sufficient data were available.

Figure 6. Between-platform agreement analysis for hERG IC50 (a) and hNav1.5 IC50 values (b). The upper right graphs display Bland-Altman plots describing between-platform agreement, with the x-axis representing mean of two determinations for each drug, and y-axis representing the difference between the two determinations (both in molar units). The lower left graphs display correlations for drugs tested across the two platforms being compared. The axes represent nominal drug test concentrations (in log units). Each dot represents one drug in the comparison datasets. p1–p6 represent five different platform with sufficient data across sites to enable comparisons.