key: cord-0862450-lthlh9g7
authors: Thomet, Urs; Amuzescu, Bogdan; Knott, Thomas; Mann, Stefan A.; Mubagwa, Kanigula; Radu, Beatrice Mihaela
title: Assessment of proarrhythmogenic risk for chloroquine and hydroxychloroquine using the CiPA concept
date: 2021-11-14
journal: Eur J Pharmacol
DOI: 10.1016/j.ejphar.2021.174632
sha: 19c4ffcd16b627796b2b83bc44aed5651743a97f
doc_id: 862450
cord_uid: lthlh9g7

Chloroquine and hydroxychloroquine have been proposed recently as therapy for SARS-CoV-2-infected patients, but during 3 months of extensive use concerns were raised related to their clinical effectiveness and arrhythmogenic risk. Therefore, we estimated for these compounds several proarrhythmogenic risk predictors according to the Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm. Experiments were performed with either CytoPatch™2 automated or manual patch-clamp setups on HEK293T cells stably or transiently transfected with hERG1, hNav1.5, hKir2.1, hKv7.1+hMinK, and on Pluricyte® cardiomyocytes (Ncardia), using physiological solutions. Dose-response plots of hERG1 inhibition fitted with Hill functions yielded IC50 values in the low micromolar range for both compounds. We found hyperpolarizing shifts of tens of mV, larger for chloroquine, in the voltage-dependent activation but not inactivation, as well as a voltage-dependent block of hERG current, larger at positive potentials. We also found inhibitory effects on peak and late I(Na) and on I(K1), with IC50 of tens of μM and larger for chloroquine. The two compounds, tested on Pluricyte® cardiomyocytes using the β-escin-perforated method, inhibited I(Kr), I(CaL), I(Na peak), but had no effect on I(f). In current-clamp they caused action potential prolongation. Our data and those from literature for I(to) were used to compute proarrhythmogenic risk predictors B(net) (Mistry HB, 2018) and Q(net) (Dutta S et al., 2017), with hERG1 blocking/unblocking rates estimated from time constants of fractional block. Although the two antimalarials are successfully used in autoimmune diseases, and chloroquine may be effective in atrial fibrillation, assays place these drugs in the intermediate proarrhythmogenic risk group.

Chloroquine and hydroxychloroquine are two classical quinoline antimalarials derived from quinine, extracted by indigenous inhabitants of South America from the bark of Cinchona officinalis and used for centuries as a remedy for chills and common cold. 

We used for experiments HEK293T cell lines stably or transiently expressing human cardiac ion channel subunits genes: stably expressed hERG1, inducible hNav1. For exploring effects on hERG1 we applied multiple voltage-clamp protocols. In both automated and manual patch-clamp experiments we applied a standard 2-step voltage-clamp protocol, starting from a holding potential of -70 mV, consisting of a 2-s depolarizing step at +40 mV followed by a 2-s step at -50 mV; the peak hERG1 current during this second step was measured relative to the average current during a brief (100 ms) step at -50 mV applied prior to the first depolarizing step.

In manual patch-clamp experiments performed on hERG1 channels at RT one single concentration of drug was tested per experiment. The standard protocol was applied under control conditions in the absence of drug and when the inhibitory effect of drug reached a steady level. In between we applied a modified Milnes protocol (Milnes et al., 2010) with 10-s depolarizing pulses at 0 mV from a holding potential of -80 mV repeated at 25-s intervals.

Membrane resistance was monitored with brief (100 ms) pulses to -90 mV applied before the main depolarizing pulses. After testing for the stability of current level at the end of the 10-s depolarizing step in control conditions drug application started. Current traces recorded during this step initially and at steady-state inhibition were used to compute the fractional block In automated patch-clamp experiments the standard voltage-clamp protocol was applied at 10-s intervals with monitoring and continuous plotting of peak hERG1 current, measured relative to the average current during the brief step at -50 mV preceding the main 2-s depolarizing step. After stabilization of peak current amplitude in control conditions a drug was applied consecutively at 4 progressively increasing concentrations under continuous perfusion for 4 min. at each concentration to monitor inhibitory effects on peak hERG current, followed by a 5-min. wash-out period to assess reversibility of drug inhibition. In a series of experiments we applied a supplementary sequence of 5 voltage-clamp protocols adapted from (Vandenberg et al., 2012) in control conditions and at each drug concentration to explore pharmacological effects on kinetics of hERG current deactivation, inactivation, recovery from inactivation, and voltage dependence of activation and inactivation. All these protocols started from a holding potential of -80 mV. For the deactivation protocol, a 500-ms depolarizing step at +40 mV was followed by 2-s steps between -50 and -160 mV in 10-mV decrements; the current traces during these steps were fitted with double exponential functions to obtain time constants of fast and slow deactivation. For the inactivation protocol, a 500-ms depolarizing step at +40 mV was followed by a brief (25 ms) step at -90 mV for fast recovery from inactivation, and then by 200-ms depolarizing steps between -80 and +60 mV in 10-mV increments, during which the time constants of hERG current inactivation were obtained by monoexponential fits. For the recovery-from-inactivation protocol, a 500-ms depolarizing step at +40 mV was followed by 500-ms steps between +40 and -160 mV in 10-mV decrements, during which the time constants of hERG current recovery from inactivation were obtained by monoexponential fits. For the voltage dependence of activation protocol, 5-s depolarizing steps between -70 and +50 mV in 10-mV increments were followed by a 300-ms step at -50 mV during which the peak hERG current was measured relative to the average level of the first J o u r n a l P r e -p r o o f sweep (with prepulse at -70 mV, where no hERG activation occurs; the current values were normalized by dividing peak currents of all sweeps with the highest peak current and plotted against the voltages of the preceding step. For the voltage dependence of inactivation protocol, a 500-ms depolarizing step at +30 mV was followed by brief (30-ms) steps between +30 and -140 mV in 10-mV decrements, during which channels were removed from inactivation to variable degrees, and then by a second 500-ms depolarizing step at +30 mV; the peak tail currents measured at the beginning of this second depolarizing step were plotted against the voltage of the preceding step, fitted with Boltzmann charge-voltage functions, then current values were normalized relative to the upper plateau value of the fit, replotted and refitted with the same function over a range of voltages where deactivation during the 30-ms step exerted negligible effects.

For the cardiac voltage-dependent Na + current component hNav1.5 we used a series of voltage-clamp protocols as follows. The protocol for peak and late INa measurement in the presence of ATX-II started from a holding potential of -120 mV, included a 677-ms preconditioning step at -130mV followed by a 33ms activation step at -20mV, a 188ms step at 0mV and a 88-ms ramp to -110 mV; the repeat interval was 7400 ms. The activation protocol started from a holding potential of -110mV, included a 677-ms preconditioning step at -130 mV followed by a 33-ms activation step between -100 mV and 0 mV in 5-mV increments (21 sweeps); the intersweep interval was 1400 ms. The inactivation protocol started from a holding potential of -110 mV, included a 677-ms preconditioning step at -140 mV up to -40 mV in 5-mV increments (21 sweeps) followed by a 33-ms activation step at -20 mV; the intersweep interval was 1400 ms.

For the slow delayed rectifier K + current hKv7.1+hMinK we used a double-step voltage-clamp protocol starting from a holding potential of -110 mV, including a 3300-ms activation step at +20mV followed by a 4400-ms step at -40mV for measurement of tail current; the repeat interval was 20000 ms.

For the cardiac inward rectifier K + current hKir2.1, transfected cells were selected based on green fluorescence via epifluorescence microscopy with blue light excitation and approached by whole-cell patch-clamp. A standard double-ramp voltage protocol, composed of a 2-s ascending ramp from -120 mV to +80 mV followed by a symmetrical descending ramp, starting from a holding potential of -70 mV, was applied repeatedly at 10-s intervals.

The current level was measured at -120 mV at the beginning of the ascending ramp. After verifying current level stability in control conditions, 3 M chloroquine or 10 M hydroxychloroquine were applied until current inhibition reached a steady value, followed by BaCl2 1 mM. The current level in BaCl2 was subtracted from previous current measurements to assess the specific hKir2.1 current component. 

Both chloroquine and hydroxychloroquine exerted strong inhibitory effects on peak hERG current amplitude measured with the standard manual two-step hERG voltage-clamp protocol (Figure 1 a,b) . During the first 2-s depolarizing step at +40 mV hERG channels open and then inactivate, while during the second 2-s step at -50 mV the channels exit the inactivated state very quickly (in a few milliseconds), giving rise to a specific tail peak hERG current. Measurements of hERG peak current amplitude were done relative to the current level during the 100-ms step at -50 mV preceding the main depolarizing step. Similar results were obtained using the same voltage protocol in automated patch-clamp experiments (CytoPatch™2) in the whole-cell configuration on HEK293T cells stably expressing hERG1. We also analyzed the voltage dependence of block induced by the two antimalarial drugs, by measuring the unblocked current following long (5 s) depolarizing steps at different potentials (as shown in Figure S2 of Supplemental files), considering only those potentials where hERG channel activation is quasicomplete. By fitting these relative peak hERG current vs. voltage plots (illustrated in Figure 3 ) with a special Boltzmann charge-voltage function (Amuzescu et al., 2003) :

where IB represents the current amplitude in the presence of the blocker, I the initial current amplitude in control conditions, Gbind the Gibbs free energy difference between blocker bound to site and free blocker in solution (neglecting transmembrane electrical field effects), V the transmembrane potential, z the valence of charged blocker molecule expressed in elementary charges (+2 for both chloroquine and hydroxychloroquine at normal pH), RT/F the "quantum" of thermal energy expressed in electrical potential units (~25.7 mV at 25°C), we could estimate relative electrical distances of the blocking site in transmembrane electrical field (a concept first proposed by Ann M. Woodhull (Woodhull, 1973) ) from the interior of the membrane  for the two compounds: 0.28 for chloroquine and 0.42 for hydroxychloroquine ( Figure 3 ).

Beyond the main blocking effects, we also explored the influence of the two antimalarials on kinetics and voltage dependence of activation and inactivation of hERG1 For studying the voltage dependence of inactivation of hERG1 channels ( Figure S3 of Supplemental files) we applied a 500-ms depolarizing step at +30 mV, followed by 35-ms steps at different hyperpolarized potentials, where hERG channels inactivated during the previous step recovered from inactivation in a voltage-dependent manner; the level of recovery from inactivation was assessed by measuring the peak tail current during the third voltage step at +30 mV. These peak tail current levels are easier to interpret than the peak currents during the preceding hyperpolarizing steps because they are elicited at the same voltage and thus directly reflect levels of hERG conductance activated during the previous step at different voltages. To obtain the voltage-dependent inactivation the peak tail currents were plotted against voltage of preceding step. The plot was fitted with a Boltzmann function, then current We also explored in separate experiments without ATX-II the effects of the two compounds on voltage dependence of activation and inactivation, and found slight but statistically significant hyperpolarizing shifts (less than 10 mV at highest concentration of drug) of half-inactivating potential V1/2, as shown in Figures S7-S8 and Tables S15-S16 of shifts produced only small effects on these changes did not seem to significantly affect simulated APs and Qnet values (Table S31- ]. In both series of simulations we used the experimental pharmacological inhibition data for chloroquine and hydroxychloroquine exposed in Table 1 , performing 1000 2-s simulation cycles for drug concentrations in the range of 0-25x Cmax. The results of these IC50-based hERG inhibition simulations were very similar, as shown in Table S31 -S32 and Figure S10 -S11. However, using the experimentally measured hERG blocking/unblocking rate modeling approach resulted in higher Qnet values, which became similar to the hERG IC50-based predicted Qnet values for chloroquine only at high concentrations (>20x Cmax, Figure S10 

In the present study we characterized inhibitory effects of chloroquine and hydroxychloroquine on human cardiac ion channels in heterologous expression systems and hiPSC-CM preparations. Assessment of inhibitory potency of these two compounds yielded IC50 values largely similar with previous reports (Table 3) Drug trapping in the inner cavity by hERG channel closure has been also described (Carmeliet, 1993 We found ( Figure 6 and Tables S9-S12 of Supplemental files) that both compounds 

In 

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This study was funded from Competitiveness Operational Programme 2014-2020 project