3.1 Patch-clamp studies of single channel characteristics
The first series of experiments assessed the effects of HCQ (Fig. 1) and
AZM alone (Fig. 2) and in combination (Fig. 3) on a range of human
cardiac ion channels. The experiments on HCQ and AZM themselves adopted
concentrations fully encompassing plasma levels associated with their
clinical use. The same HCQ concentration range was then combined with 10
µM AZM. These examined inward, cardiac channel currents from hNav1.5
(INa, A ), Cav1.2 (ICa,L,B ), mediating or maintaining membrane action potential
depolarization. They also studied outward Kv4.3 (Ito,C ), hERG (IKr, C ), KCNQ1/E1
(IKs, E ) and Kir2.1 (IK1,F ) normally mediating action potential recovery. This encompassed
the range of ion channels recommended by the CiPA protocol.
Figure1 (left panels, i) exemplifies observed ionic currents in the
presence of 0, 1, 10 and 100 µM HCQ. The insets summarize the related
voltage clamping protocols, fully
described in extended Methods in Supplementary data. Five concentrations
ranging from 10-8 to 10-4 M were
tested for each current examined in 3 to 6 cells at each concentration.
Mean concentration-response plots (right panels, ii) used the maximum
magnitudes of each current. Where applicable, the consequent alterations
in such current magnitudes following pharmacological challenge were used
to derive IC50 values.
Figure1 (right panels, ii) plots mean (±SEM) fractional reductions in
current magnitude against HCQ concentration for INa (A),
ICaL (B), Ito (C), IKs(D), IKr (E) and IK1 (F) . µM-HCQ
inhibited both IKr tail and IK1 pulse
currents with IC50s of 10±0.6 µM (D) and 34±5 µM (F),
respectively, with more marked effects on IKr, where the
IC50 fell close to expected therapeutic levels in
current Covid-19 treatment regimes (Borba et al., 2020), than
IK1. In contrast, µM-HCQ has less effect on
INa and ICaL with IC50s
of 113 ± 78 µM and 209 ± 94 µM respectively, Nevertheless, HCQ caused
graded INa reductions through the entire HCQ
concentration range typified in a 22±1.8% reduction at 1 µM. No
significant inhibition of Ito and IKs by
µM-HCQ was observed.
Figure 2 presents corresponding results obtained with AZM applied by
itself similarly encompassing clinical therapeutic levels. A previously
reported dose regime administering 500, 250 and 250 mg/day on each of
three successive days gave a day 2 serum concentration of 0.22 μM(Lai,
Ho, Jain & Walters, 2011)). However, the entire, 0-100 μM explored AZM
concentration range yielded only minimal inhibitory effects. Even 10 µM
and 100 µM AZM reduced INa (A), ICaL,(B) IKs (E) and IKr (D) by
<17% and <35% respectively and produced negligible
effects on IK1 (F) and no effect on Ito(C).
Finally, Figure 3 summarizes results of applying the same range of five
HCQ concentrations displayed in Fig. 1 now combined with 10 µM AZM.
Adding AZM did not significantly alter the effects of HCQ on
INa (A), ICaL (B), Ito(C), and IKs (E), but reduced the
IC50 for IKr (IC50 = 7.7
± 0.8 µM) and IK1 (30.4 ± 3.0 µM).