In Vitro Cardiovascular Effects of Dihydroartemisin-PiperaquineCombination Compared with Other Antimalarials
Franco Borsini,a William Crumb,b Silvia Pace,a David Ubben,c Barb Wible,d Gan-Xin Yan,e and Christian Funck-Brentanof
Sigma-Tau Industrie Farmaceutiche Riunite s.p.a., Pomezia, Italya; Zenas Technologies LLC, Matairie, Louisiana, USAb; Medicine for Malaria Venture (MMV), Geneva,Switzerlandc; ChanTest Corporation, Cleveland, Ohio, USAd; Lankenau Institute for Medical Research, Wynnewood, Pennsylvania, USAe; and UPMC Université Paris 06,Faculty of Medicine, Department of Pharmacology and UMRS 956, Paris, France; AP-HP, Pitié-Salpêtrière Hospital, Department of Pharmacology and CIC-9304, Paris,France; and INSERM, CIC-9304 and UMRS-956, Paris, Francef
The in vitro cardiac properties of dihydroartemisinin (DHA) plus piperaquine phosphate (PQP) were compared with those ofother antimalarial compounds. Results with antimalarial drugs, chosen on the basis of their free therapeutic maximum concen-tration in plasma (Cmax), were expressed as the fold of that particular effect with respect to their Cmax. The following tests wereused at 37°C: hERG (human ether-a-go-go-related gene) blockade and trafficking, rabbit heart ventricular preparations, and so-dium and slow potassium ion current interference (INa and IKs, respectively). Chloroquine, halofantrine, mefloquine, and lume-fantrine were tested in the hERG studies, but only chloroquine, dofetilide, lumefantrine, and the combination of artemether-lumefantrine were used in the rabbit heart ventricular preparations, hERG trafficking studies, and INa and IKs analyses. A properreference was used in each test. In hERG studies, the high 50% inhibitory concentration (IC50) of halofantrine, which was lowerthan its Cmax, was confirmed. All the other compounds blocked hERG, with IC50s ranging from 3- to 30-fold their Cmaxs. In hERGtrafficking studies, the facilitative effects of chloroquine at about 30-fold its Cmax were confirmed and DHA blocked it at a con-centration about 300-fold its Cmax. In rabbit heart ventricular preparations, dofetilide, used as a positive control, revealed a highrisk of torsades de pointes, whereas chloroquine showed a medium risk. Neither DHA-PQP nor artemether-lumefantrine dis-played an in vitro signal for a significant proarrhythmic risk. Only chloroquine blocked the INa ion current and did so at about30-fold its Cmax. No effect on IKs was detected. In conclusion, despite significant hERG blockade, DHA-PQP and artemether-lu-mefantrine do not appear to induce potential torsadogenic effects in vitro, affect hERG trafficking, or block sodium and slowpotassium ion currents.
Torsade de pointes (TdP) caused by drugs is a life-threateningform of polymorphic ventricular tachycardia which is associ-
ated clinically with a long QT interval prolongation. This kind ofdeleterious effect has been described in several classes of drugs,such as antihistamines, psychotropics, and antibiotics (40). How-ever, QT prolongation is not a strong predictor of the risk of TdP,and several factors may contribute to an individual patient’s riskof TdP. Various models have been developed to assess the poten-tial in vitro cardiac toxicity of drugs and their potential to generateTdP (9). Many antimalarials are associated with prolongation ofthe corrected QT interval (QTc) at therapeutic concentrations(49). However, despite large-scale use, information on the rate ofTdP with these drugs is limited, because these drugs are mostlyused in developing countries, where pharmacovigilance data arelacking. Dihydroartemisinin (DHA) plus piperaquine phosphate(PQP) is a fixed-combination antimalarial treatment with excel-lent efficacy and good tolerance (3, 4, 14, 16, 29, 38, 46). Deriva-tives of artemisinin are generally considered to be safe in terms ofcardiotoxic potential, with no clinically significant changes in QTobserved in the treatment of malaria (49). QTc prolongation ap-pears to be limited with DHA and PQP (49). However, PQP isstructurally similar to chloroquine, for which significant electro-physiological effects on the heart have been described (43, 45),even if the clinical differences have not been well considered.
One common electrophysiological finding for those antima-larial drugs associated with QTc prolongation is blockade of therepolarizing potassium channel hERG (human ether-a-go-go-re-lated gene) (24, 35, 45). To investigate the electrophysiologicalprofile of DHA and PQP, the hERG channel-blocking profile of
these compounds, alone and in combination, was characterized instably expressing human embryonic kidney (HEK-293) cells. Forcomparison, the hERG channel-blocking effects of chloroquine,dofetilide, halofantrine, lumefantrine, and mefloquine were alsoevaluated. Previous hERG studies with antimalarials were per-formed in vitro at room temperature (24, 35, 45). Under our ex-perimental conditions, the hERG assay was evaluated at 37°C,which provides a more conservative safety evaluation of hERGinhibition (27).
The potential torsadogenic risk of DHA, PQP, and their com-bination was evaluated in a rabbit heart wedge preparation (33),an established experimental model for the prediction of drug-induced proarrhythmia (26). In this model, the torsadogenic riskscore ranges from �2 up to 14, with a higher score being worse(33). The results with the combination of DHA and PQP werecompared to those with chloroquine, artemether combined withlumefantrine, and dofetilide. Dofetilide was used as a positive con-trol because of the numerous reports of TdP associated with thismedication (1). Additionally, three in vitro tests were performedwith DHA, PQP, and their combination: hERG trafficking (50)
Received 8 September 2011 Returned for modification 24 December 2011Accepted 26 February 2012
and sodium and slow potassium ion current interference (INa andIKs, respectively) measurements (11). These tests give additionalinsights into the possible mechanism of cardiac toxicity.
The aim of the present work was to evaluate the effects of thecombination of DHA and PQP and its components on in vitrotests used to predict cardiac proarrhythmic risk.
MATERIALS AND METHODShERG. (i) Transfection and cell culture. Because rapid potassium ioncurrent interference (IKr) is regionally expressed in human heart and dif-ficult to record in native cells, the cloned equivalent of the human IKr
(hERG) was used in this study. The pharmacology of this cloned channelexpressed in a human cell line has been shown to be very similar to thatdetermined in native cardiac tissues. The hERG channel is expressed in anHEK-293 cell line that lacks endogenous hERG channels. HEK-293 cellswere stably transfected through the Lipofectamine method (42) with thehERG clone.
(ii) External and internal recording solution and components. Noexpiration date for the individual components of the external recordingsolution was given by the manufacturer. An expiration date of 3 years wasset for the individual components from the time of reception. Cells weremaintained in the following medium in culture flasks with materials pur-chased at Cellgro (Mediatech): minimum essential medium with Earle’ssalts (86.9 ml), nonessential amino acids (1 ml), sodium pyruvate (1 ml),penicillin-streptomycin (1 ml), fetal bovine serum (10 ml), and Geneticin(100 �l). The external recording solutions were made up by NaCl (137mM), KCl (4 mM), MgCl2 (1 mM), CaCl2 (1.8 mM), HEPES (10 mM),and dextrose (11 mM), adjusted to a pH of 7.4 with NaOH (Sigma). Theexternal recording solution was not used for more than 2 weeks afterpreparation. The composition of internal recording solution was made upby KCl (130 mM), MgCl2 (1 mM), HEPES (5 mM), EGTA (5 mM), andNaCl (7 mM), adjusted to a pH of 7.2 using KOH. In a second set ofexperiments, Na-ATP (5 nM) was added to the internal recording solu-tion (see below).
(iii) Experimental methods. Experiments were performed at 37 �1°C. Currents were measured using the whole-cell variant of the patchclamp method. Glass pipettes were pulled from borosilicate glass by ahorizontal puller (Sutter Instruments) and then fire polished to producetip openings of 1 to 2 �m. Pipette tip resistance was approximately 1 to 2M� when the pipette was filled with internal recording solution. Bathtemperature was measured by a thermistor placed near the cell understudy and was maintained by a thermoelectric device (model no. 806-7243-01; Cambion/Midland Ross, Cambridge, MA). An Axopatch 1-Bamplifier (Axon Instruments, Foster City, CA) was used for whole-cellvoltage clamping. Creation of voltage clamp pulses and data acquisitionwere controlled by a personal computer (PC) running pClamp software(Axon Instruments). From a holding potential of �75 mV, preparationswere depolarized at �10 mV for 500 ms and repolarized to �40 mV foranother 500 ms, before they were returned to the holding potential. Afterrupture of the cell membrane (entering whole-cell mode), current kineticsand amplitudes were allowed to stabilize as the cell was dialyzed withinternal recording solution and stimulated at 0.1 Hz using the protocoldescribed below. Current kinetics and amplitude typically stabilized inapproximately 5 min. If the hERG current did not stabilize over this timeperiod, the cell was discarded. Currents were considered stable if currentselicited by a series of voltage pulses given at 0.1 Hz were superimposable.Peak hERG current was measured as the maximum outward deflection ofthe tail current elicited upon return to �40 mV. In all experiments, drugwas added in a cumulative manner.
Means � standard errors of the means (SEMs) are given. Analyzeddata are presented as the percent reduction of the current amplitude aftera steady-state effect was reached in the presence of drug relative to thecurrent amplitude before the test substance was superfused (control).Each cell served as its own control. Test substance effects were comparedby a paired Student t test for significance (P � 0.05) using MicroCal
Origin, version 6.0, software. Log-linear plots of the mean percent block-ade � SEM at the concentrations tested were created. A nonlinear curve-fitting routine was utilized to fit a three-parameter Hill equation to theresults using MicroCal Origin, version 6.0, software. The equation is y �Vmax[xn/(kn � xn)], where y is the IC50, x is the concentrations, Vmax
(which was equal to 100) is the maximum rate of metabolism, and k and nare unconstrained variables.
The experiment with DHA and PQP was repeated twice with differentconcentrations: six concentrations on five cells on the first set and three tofive concentrations on four to five cells on the second set.
hERG trafficking studies. The objective of the hERG trafficking stud-ies was to obtain a rapid estimate of the in vitro effects of test articles onsurface expression of the wild-type (WT) hERG (human ether-a-go-go-related gene) potassium channel. The cardiac potassium channel, hERG,is responsible for the rapid delayed rectifier current (IKr) in the humanventricle. This channel is selected because inhibition of IKr is the mostcommon cause of cardiac action potential prolongation by noncardiacdrugs (5, 48, 55). In addition to direct block, drug-induced traffickinginhibition of hERG has been linked to QT prolongation (18, 30). In-creased action potential duration causes prolongation of the QT intervalthat has been associated with ventricular arrhythmia, or torsade depointes.
(i) Cell culture procedures. HEK-293 cells were transfected withhERG WT cDNA. Stable transfectants were selected by coexpression ofthe hERG cDNA and G418 resistance gene incorporated into the expres-sion plasmid. Selection pressure was maintained by including G418 in theculture medium. Cells were cultured in Dulbecco’s modified Eagle medi-um–nutrient mixture F-12 (DMEM/F-12) supplemented with 10% fetalbovine serum, 100 U/ml penicillin G sodium, 100 �g/ml streptomycinsulfate, and 500 �g/ml G418. Cells were maintained in tissue culture in-cubators at 37°C in a humidified 95% air, 5% CO2 atmosphere, withstocks maintained in cryogenic storage. Cells for HERG-Lite assays wereplated in 96-well microplates.
(ii) Experimental methods. Cells were incubated with a test article(s)overnight (minimum of 16 h) in a tissue culture incubator at 37°C in ahumidified 95% air, 5% CO2 atmosphere. To assay hERG surface expres-sion, cells were fixed with freshly prepared paraformaldehyde. Nonspe-cific antibody binding sites were blocked by incubation in 1% goat serum–phosphate-buffered saline. The cells were sequentially incubated with anantibody against an extracellular epitope incorporated into the hERGchannel and a secondary antibody coupled to a light-generating enzyme.A DNA-binding fluorescent dye was added to the secondary antibodysolution to monitor cell number. The fluorescent signals were captured ina microplate fluorescence reader, after which the chemiluminescent re-agent detection mix was added to the cells and the luminescent signalswere detected in a microplate luminometer.
Since some test articles may be toxic, a DNA-binding fluorescent dyewas used in the HERG-Lite protocol to determine the cell number per wellat the end of the experiment. A standard curve of fluorescence versus cellnumber was performed. Chemiluminescence values were corrected forcell loss up to 75%. If cell loss exceeded 75%, the test article was deemedtoo toxic at that concentration to obtain reliable data.
The mean surface expression (relative chemiluminescence units) ofthe test article wells was compared to the surface expression (mean �standard deviation) of the control wells. A significant change in surfaceexpression produced by the test article is indicated if the mean of the testarticle wells is 3 standard deviations or more away from the mean of thevehicle control. The surface expression changes were compared to those inthe presence of the positive-control article.
Rabbit heart ventricular wedge preparations. New Zealand Whitefemale rabbits weighing 2.2 to 3.0 kg were anticoagulated with heparinand anesthetized by endovenous injection of ketamine-xylazine (40 to 50mg/0.5 to 1.0 mg per kg of body weight). The chest was opened via a leftthoracotomy, and the heart was excised and placed in a cardioplegic so-lution consisting of cold (4°C) normal Tyrode’s solution. Transmural
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wedges with dimensions of approximately 1.5 cm in width and 2 to 3 cmin length were dissected from the left ventricle as described previously(33). The tissue was cannulated via the small branch of the left anteriordescending artery and perfused with cardioplegic solution. The prepara-tion was then placed in a small tissue bath and arterially perfused withTyrode’s solution containing 4 mM K� buffered with 95% O2 and 5%CO2 (temperature, 35.7 � 0.1°C; perfusion pressure, 40 to 50 mm Hg).The ventricular wedge was allowed to equilibrate in the tissue bath untilelectrically stable for 1 h. The preparations were stimulated at basic cyclelengths (BCLs) of 1,000 and 2,000 ms using bipolar silver electrodes insu-lated except at the tip and applied to the endocardial surface.
(i) Electrophysiological recordings. A transmural electrocardio-graphic (ECG) signal was recorded via an HP ECG amplifier (model8811A) using extracellular silver/silver chloride electrodes placed inTyrode’s solution, bathing the preparation 1.0 to 1.5 cm from the epicar-dial and endocardial surfaces, along the same vector as the transmem-brane recordings. The QT interval was defined as the time from the onsetof the QRS to the point at which the final downslope of the T wave inter-sects with the isoelectric line. Transmembrane action potential from theendocardium (endo) was recorded at a BCL of 2,000 ms via a customer-made amplifier. Transmural dispersion of repolarization (TDR) was de-fined as the interval between the end (e) and the peak (p) of the T wave(Tp-e) (53, 54). All measured biological signals, including ECG and trans-membrane action potentials, were sampled via a digital-to-analog con-verter (1401; Cambridge Electronic Design [CED], England) and storedon electronic media (compact disk). The raw signals of ECG and trans-membrane action potentials were analyzed using Spike 2 software (CED,England).
(ii) Experimental methods. Each compound or combination exceptDHA (2.4 �M)-PQP was tested at five concentrations in five wedge prep-arations; for DHA (2.4 �M)-PQP, six concentrations were tested in fourpreparations.
Each preparation was exposed to each compound or combination ateach concentration for �30 min (Fig. 1). Two BCLs of 1,000 and 2,000 mswere used. Action potentials from endo, the QT and QRS intervals, andthe interval Tp-e, an index of TDR, were measured at a BCL of 2,000 ms.Arrhythmic phenomena, including spontaneous early after depolariza-tion (EAD), R-on-T ectopic beats, and TdP, were recorded if they oc-curred. The combination of these events originated the torsadogenic riskscore (Table 1).
Data were analyzed using one-way analysis of variance (if data wereparametric) or the Friedman test (if data were not parametric) for re-peated measurements and then Holm-Sidak’s or Dunn’s method as posthoc statistical analysis.
INa and IKs currents. (i) Isolation of cardiac myocytes. Human myo-cytes were obtained from specimens of human right atrial appendageobtained during surgery from hearts of patients undergoing cardiopul-monary bypass (12). Samples were quickly immersed in a cardioplegicsolution consisting of 50 mM KH2PO4, 8 mM MgSO4, 10 mM NaHCO3,
5 mM adenosine, 25 mM taurine, 140 mM glucose, and 100 mM manni-tol, titrated to a pH of 7.4, and bubbled with 100% O2 at 0 to 4°C. Speci-mens were minced into 0.5- to 1-mm cubes and transferred to a 50-mlconical tube containing an ultra-low-calcium wash solution containing137 mM NaCl, 5 mM KH2PO4, 1 mM MgSO4, 10 mM taurine, 10 mMglucose, 5 mM HEPES, and 100 �mol/liter (�M) EGTA, pH 7.4 (22 to24°C). The tissue was then gently agitated by continuous bubbling with100% O2 for 5 min. The tissue was next incubated in 5 ml of solutioncontaining 137 mM NaCl, 5 mM KH2PO4, 1 mM MgSO4, 10 mM taurine,10 mM glucose, and 5 mM HEPES supplemented with 0.1% bovine albu-min, 2.2 mg/ml collagenase type V, and 1.0 mg/ml protease type XXIV(Sigma Chemical), pH 7.4 (37°C), and bubbled continuously with 100%O2. The supernatant was removed after 20 min and discarded. The chunkswere then incubated in a solution of the same ionic composition butsupplemented with only collagenase and 100 �M CaCl2. Microscopic ex-amination of the medium was performed every 5 to 10 min to determinethe number and quality of the isolated cells. When the yield appeared to bemaximal, the cell suspension was centrifuged for 2 min and the resultingpellet was resuspended in a modified Kraftbruhe solution containing 25mM KCl, 10 mM KH2PO4, 25 mM taurine, 0.5 mM EGTA, 22 mM glu-cose, 55 mM glutamic acid, and 0.1% bovine albumin, pH 7.3 (22 to24°C). In general, the isolation procedure produced an initial yield ofapproximately 40 to 60% rod-shaped, calcium-tolerant cells. Cells wereused within 24 h after isolation.
(ii) Experimental methods for INa and IKs. Experiments were per-formed at 36 � 1°C. Currents were measured using the whole-cell variantof the patch clamp method. Glass pipettes were pulled from borosilicateglass by a horizontal puller (Sutter Instruments) and then fire polished toproduce tip openings of 1 to 4 �m. Pipette tip resistance was approxi-
FIG 1 Experimental protocol used to study the test article in the isolated arterially perfused rabbit ventricular preparations. The data recording (R) is made 30to 60 s before the end of each stimulation period. Control, control perfusion.
TABLE 1 Score system for estimate of risk of a compound for relativeTdP risk using the isolated rabbit left ventricular wedge preparationa
ScoreDifference in QTinterval (%) Tp-e/QT ratio (%) Phase 2 EAD
�1 ��5 ��50 �5–�10 �5–�101 10–�20 10–�202 20–�30 20–�30 �EAD3 �30 �304 EAD without R on T6 EAD with R on T8 TdPa Points for the QT interval, the Tp-e/QT ratio, and phase 2 EAD are provided (7). Themaximal score is 14, and the minimum is �2. Basic cycle lengths (BCLs) are 2,000 ms.EAD, early after depolarization; �EAD, equivocal EAD from endocardial actionpotential at a BCL of 2,000 ms when the QT interval is �30%; TdP, torsade de pointes;Tp-e, interval between the end (e) and the peak (p) of the T wave; QT, QT interval.
mately 1.0 to 2.0 M� when filled with internal solutions. Bath tempera-ture was measured by a thermistor placed near the cell under study. AnAxopatch 1-B amplifier (Axon Instruments, Foster City, CA) was used forwhole-cell voltage clamping. Creation of voltage clamp pulses and dataacquisition were controlled by a PC running pClamp software (version9.2; Axon Instruments).
After rupture of the cell membrane (entering whole-cell mode), cur-rent kinetics and amplitudes were allowed to stabilize as the cell was dia-lyzed with internal solution and paced at 0.1 Hz (typically, 3 to 5 min). Ifthe current did not stabilize over this time period, the cell was discarded.Currents were considered stable if currents elicited by a series of voltagepulses given at 0.1 Hz were superimposed.
INa was elicited by a pulse to �20 mV from a holding potential of �120mV (pulse duration, 40 ms). Peak inward current was measured for INa.IKs was elicited by a 5-s voltage pulse to �10 mV from a holding potential
of �40 mV. A pacing rate of 0.1 Hz was used for both ion currents. Drugswere added in a cumulative manner; i.e., 0.1 �M was added to a cell untilsteady state was reached, generally in 2 to 3 min, then 0.3 �M, and so on.
Drugs. With the exception of PQP (Sigma-Tau Industrie Farmaceu-tiche Riunite, Rome, Italy), which was dissolved in water, all the othercompounds, artemether (Tokyo Chemical Industry Co., Tokyo, Japan),chloroquine (Sigma-Aldrich, Milan, Italy), dofetilide (Sigma-Aldrich,Milan, Italy), dihydroartemisinin (DHA; Sigma-Tau Industrie Farmaceu-tiche Riunite, Rome, Italy), halofantrine (Sigma-Aldrich, Milan, Italy),lumefantrine (LGM Pharma Inc., Boca Raton, FL), and mefloquine(Sigma-Aldrich, Milan, Italy), were dissolved as stock solutions in di-methyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) at a maximumof 1% (vol/vol). Then, stock solutions were vortexed until the solutionwas clear and stored in a freezer, whose temperature ranged from �19 to�22°C. DMSO and geldanamycin were purchased from Sigma-Aldrich(St. Louis, MO). Drug concentrations were chosen to cover a wide rangeof subtherapeutic, therapeutic, and supratherapeutic concentrations us-ing the estimated free fraction around the maximum concentration inplasma (Cmax) in humans as the therapeutic concentrations, as indicatedin Table 2.
RESULTShERG currents. The various compounds decreased the hERGcurrent in a concentration-related manner (Fig. 2). The combina-tion of DHA at two concentrations (2.4 �M and 7.2 �M) in thepresence of different concentrations of PQP produced a level ofhERG block which was apparently less than that produced by PQPalone and somewhat less than that produced by DHA alone (Table3). Historically, this channel in our lab is blocked by E-4031 (21,22, 44), a selective and potent hERG blocker, with a 50% inhibi-tory concentration (IC50) of 18.1 nM (n � 6 to 11). In 3 cells,current rundown and the effects of the vehicle (DMSO) were as-sessed by monitoring current when cells were exposed to DMSO
TABLE 2 Drugs used for the various studies with references and totaland free plasma concentrations in patients
a Data are in nM for dofetilide.b SmPC, summary of product characteristics.c The datum was obtained from single-dose Cmax by applying an accumulation factorof 2.5.
FIG 2 Effects of various antimalarial drugs on hERG current at 0.1 Hz. Five concentrations of drugs were used, and concentrations ranged from 0.003 to 0.3 nMfor halofantrine, from 0.058 to 5.8 �M for PQP, and from 0.3 to 30 �M for lumefantrine (LUM) and DHA. Six concentrations were used for chloroquine andmefloquine, and concentrations ranged from 0.1 to 30 �M. Five different cells were used for each concentration. The actual IC50s are reported in the inset. Valuesrepresent mean � SEM of five different cells.
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(1/300, vol/vol) over the time period of a typical experiment (6 to8 min). In two cells, current was decreased by 2.7% and 3.1%, andin the other cell it was increased by 1.7%. This indicates that in theabsence of test article there was no substantial reduction in hERGcurrent amplitude.
hERG trafficking. The only test article that produced signifi-cant inhibition of hERG trafficking was DHA at 100 �M (Fig. 3).However, cells in one vial with such a DHA concentration had amortality rate higher than 75%, and therefore, the result from thisvial was not considered. In contrast, chloroquine increased hERGtrafficking, as already reported (50). The test system worked prop-erly, as geldanamycin (1 �M), the positive-control trafficking in-hibitor for WT hERG, produced the expected result with a 50%decrease in WT hERG surface expression (50).
Rabbit ventricular wedge preparations. The system, i.e., theisolated arterially perfused rabbit left ventricular wedge prepara-tion, was validated by using dofetilide, which produced concen-tration-dependent torsadogenic risk effects, starting from 0.3 nM(Fig. 4; Table 4). Moreover, prolonging effects on QT, Tp-e, andendocardial action potential duration (APD) measured at 90%repolarization (endo-APD90) were also observed at 3 nM (Table4), and EADs were evident in four out of five preparations at 10nM and in all five preparations at 30 nM.
DHA had a mild negative effect on potential torsadogenic risk(Fig. 4; Table 4), which was statistically observed only at 100 �M
(but see Discussion). PQP increased the QT interval, Tp-e, andendo-APD90 at 0.1 to 1 �M, but no arrhythmic events were ob-served (Table 4). PQP increased the TdP risk score only at 3 �M,i.e., more than 100-fold its Cmax. When DHA (at 2.4 and 7.2 �M)and PQP were coadministered, the TdP risk scores were notchanged (Table 4), even if PQP was used at 10 �M (Table 4). Alsoin such a case, no EADs were observed. Lumefantrine did notinduce TdP (Table 4); however, it significantly increased QT from0.3 to 10 �M and APD90 at 10 �M (Fig. 4; Table 4). No EAD wasobserved. Likewise, the association of artemether and lumefan-trine did not modify such effects (Table 4).
Chloroquine induced mild TdP risk scores at concentrations of�3 �M, at which QT and endocardial APD90 were significantlyincreased (Table 4). Chloroquine induced one EAD out of fivepreparations at 10 �M and at such a concentration increased theQRS interval.
INa current. All of the compounds tested produced less than a50% inhibition of the human cardiac sodium current (Fig. 5). Theblock of chloroquine (42.6%) is in the same range of the values(45%) already published for feline ventricular myocytes (43).
IKs current. The IKs-blocking profiles of DHA, PQP, the DHAand PQP combination, chloroquine, and dofetilide (Fig. 6) pro-duced less than 20% inhibition. In one cell, 50 �M chromonal293B, a selective IKs blocker (15), was added to the bath solution.In this cell, IKs was blocked by 98.3%, confirming cell sensitivity.
DISCUSSION
The in vitro concentrations of nonclinical cardiac toxicity experi-ments should be consistent with the free therapeutic plasma con-centrations (33, 41). For the present analyses, free therapeuticplasma concentrations were used, as reported in Table 2, to deter-mine the central concentrations around which sub- and suprath-erapeutic concentrations were examined.
Consistent with the clinically observed QT prolongation, DHAcombined with PQP and lumefantrine blocked IKr channel cur-
TABLE 3 IC50s on hERG current at 0.1 Hz by DHA and PQP and theircombination in a second set of experiments
Compound IC50 (�M) � SEMa
DHA 7.7 � 0.9PQP 0.087 � 0.0032.4 �M DHA � PQP 0.201 � 0.0017.2 �M DHA � PQP 0.179 � 0.008a Values represent the mean � SEM of 4 to 5 cells.
FIG 3 Effects of various antimalarial drugs on hERG trafficking. Five concentrations of drugs were used, and concentrations ranged from 0.003 to 3 �M for PQP(with or without 2.4 or 7.2 �M DHA), from 1 to 100 �M for DHA, from 0.1 to 10 �M for chloroquine, from 0.3 to 30 �M for lumefantrine (LUM; with 0.18 �Martemether [ART]), and from 0.1 to 10 nM for dofetilide. Geldanamycin was given at 1 �M (black spot). Values represent mean � SEM of five different cells.
rents (hERG). The effects of PQP and DHA on hERG currentswere quite stable, as they ranged, in the two different experiments,from 0.106 to 0.087 �M for PQP and 9.62 to 7.7 �M for DHA.
The IC50s on hERG are similar but somewhat more potent thanthose previously reported. For example, the IC50 for halofantrinehas been reported to range from 21.6 to 40 nM (35, 45), while thepresent study yielded a value of 18 nM. Chloroquine, mefloquine,and lumefantrine have previously been reported to have IC50s of2.5, 2.6 to 5.6, and 8.1 �M, respectively (25, 35, 45), while thepresent experiment yielded values of approximately 1.0, 1.0, and2.6 �M, respectively. The increased potency in the present study ismost likely explained by the use of physiologic temperatures(37°C) versus room temperature in previous studies. Increasedpotency at elevated temperatures has been reported for otherdrugs as well, such as erythromycin and sotalol (27, 51). The com-bination of DHA at two concentrations (2.4 and 7.2 �M) in thepresence of PQP was also evaluated, and hERG blockade by DHA-PQP was less than that by PQP alone. However, retrospectiveanalysis of the risk of TdP and the ability of a drug to inhibit thischannel demonstrates that some compounds already on the mar-ket can block this channel without inducing TdP (6, 41). It isgenerally accepted that hERG blockade can predict clinical QTprolongation in many cases but not TdP (19, 34, 40). It should,however, be acknowledged that QT prolongation is found in hu-mans at estimated free concentrations of PQP which are 10 timeslower (0.008 �M) than those which block 50% of hERG activity invitro (0.087 �M). A possible explanation for this discrepancy isthat QT prolongation in vivo may occur at levels of hERG block-ade in the 10% range (23).
DHA exerted blocking of hERG trafficking effects only at aconcentration that induced cell deaths (�75%) in 1 out of 6 vials.This result is not surprising, as it has already been shown that
DHA may already increase apoptosis at �M concentrations afterseveral hours of incubation (31, 36, 56). It is worth mentioningthat the execution of the hERG trafficking test requires 16 h ofincubation with the test compounds. As already reported, chloro-quine increased hERG trafficking (50). Thus, it seems that theeffect of hERG blockade by the various antimalarial drugs in-volved increased protein hERG synthesis only by chloroquine.
The electrophysiological properties of the isolated rabbit ven-tricular wedge preparation are integrative functions of major de-polarization and repolarization ion currents such as INa, IKr, andIKs (33). As also shown by the dedicated studies, neither DHA-PQP nor artemether-lumefantrine significantly affected Na orslow K currents. The lack of interaction with fast INa is also dem-onstrated by the absence of QRS prolongation, as reported, forexample, for the Na channel blocker flecainide (28). The heartwedges are considered a sensitive and specific experimental modelmimicking human pathological conditions characterized by asubstantial reduction in repolarization reserve (24). These condi-tions (hypokalemia, bradycardia, etc.) are recognized risk factorsfor the potential of QT-prolonging agents to trigger TdP, a life-threatening arrhythmia. Similarly, the Tp-e interval describestransmural dispersion of repolarization that plays an importantrole in the development of TdP (53, 54). Moreover, the Tp-e/QTratio provides important electrophysiological information abouta drug (33): (i) when there is QT interval prolongation, a concur-rent increase in the Tp-e/QT ratio signals strong IKr blockade be-cause IKr blockade is accompanied by a preferential prolongationof endocardial action potential in the rabbit ventricular wedgepreparation (33, 54); and (ii) when there is QT interval prolonga-tion, an unchanged or decreased Tp-e/QT ratio signals IKs blockadeor combined IKr and INa blockade if the QRS duration increases(33) or signals combined IKr and calcium current interference
FIG 4 Effects of various antimalarial drugs on TdP risk score in comparison with those of dofetilide. Values represent mean � SEM of five preparations.Concentrations of drugs ranged from 0.003/0.1 to 3/10 �M for PQP without or with 2.4 or 7.2 �M DHA, from 1 to 100 �M for DHA, from 0.1 to 10 �M forchloroquine, from 0.3 to 30 �M for lumefantrine (LUM; without or with 0.18 �M artemether [ART]), and from 0.3 to 30 nM for dofetilide. Horizontal dashedlines represent scores of 1 and 7.25, which are considered to be associated with moderate and marked risks of torsades de pointes, respectively, when they arereached at concentrations less than 100 the free therapeutic plasma Cmax (32).
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a All values represent mean � SEM of five preparations (except for 2.4 �M DHA plus 10 �M PQP, where only four preparations were used). Endo, endocardium; *, P � 0.05 versus controlgroup; **, P � 0.02 versus control group; ***, P � 0.01 versus control group; ND, no valid data available because the compound was not completely dissolved in the Tyroide’s solution.
(ICa,L [where L is L-type calcium current]) blockade (47). Proar-rhythmic events measurable in the rabbit ventricular wedge prep-aration include EAD as well as EAD-dependent phenomena (i.e.,R-on-T extrasystole and TdP), ventricular tachycardia, and ven-tricular fibrillation. It is widely accepted that TdP is initiated byEAD-dependent R-on-T extrasystoles under conditions of QTprolongation (24, 54). EADs as well as EAD-dependent R-on-Textrasystoles and TdP often occur in the presence of potent IKr
blockers in the rabbit ventricular wedge preparation (24, 33, 54).
Therefore, the use of this parameter significantly reduces theprobability of false positives by increasing the specificity of thepreclinical study (24). On the other hand, strong use-dependentblockade of the fast sodium channel can result in monomorphicventricular tachycardia and increase mortality in patients withcoronary artery disease and left ventricular systolic dysfunction(8, 20).
However, DHA-PQP and artemether-lumefantrine, despitetheir hERG blockade, did not induce proarrhythmic events, as
FIG 5 Effects of various antimalarial drugs on INa ion current. Values represent mean � SEM of five preparations. Five or six concentrations of drugs were used,and the concentrations ranged from 0.003/0.1 to 3/10 �M for PQP without or with 2.4 or 7.2 �M DHA, from 1 to 100 �M for DHA, from 0.1 to 10 �M forchloroquine, from 0.3 to 30 �M for lumefantrine (LUM; with 0.18 �M artemether [ART]), and from 0.1 to 10 nM for dofetilide.
FIG 6 Effects of various antimalarial drugs on IKs ion current. Values represent mean � SEM of five preparations. Five or six concentrations of drugs were used,and the concentrations ranged from 0.003/0.1 to 3/10 �M for PQP without or with 2.4 or 7.2 �M DHA, from 1 to 100 �M for DHA, from 0.1 to 10 �M forchloroquine, from 0.3 to 30 �M for lumefantrine (LUM; with 0.18 �M artemether [ART]), and from 0.1 to 10 nM for dofetilide.
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shown by any effect on the TdP risk score. In the case of lumefan-trine, however, the difficulty in dissolving it at 30 �M was noticedwhen it was to have been infused into the wedge preparation.Thus, the effects with lumefantrine at 30 �M should be inter-preted with caution, both in the hERG and hERG trafficking prep-arations and in the INa and IKs current studies. The TdP risk scorewas increased by chloroquine and was very clearly increased bydofetilide, with the latter inducing EADs in all five heart wedgepreparations. These observations with dofetilide were never re-ported and confirm the predictivity of the test.
The aim of the present studies was to evaluate the potentialarrhythmogenic effects of DHA-PQP. Despite the experimentallimits (cytotoxicity at high DHA concentration, difficulty in dis-solving 30 �M lumefantrine), our experiments suggest that bothDHA-PQP and artemether-lumefantrine may have a low poten-tial to induce TdP in the rabbit ventricular wedge model. Usingthis model, both combination compounds appear to be less activein generating a risk of TdP than chloroquine. Chloroquine hasbeen reported to induce heart rate-corrected QT (QTc) prolonga-tion between 14 and 30 ms (8, 10). A few reports (2, 13, 17, 39)describe an event consistent with TdP ventricular arrhythmia withchloroquine; however, there is no sign of sudden unexplaineddeath associated with its use as an antimalarial. These results com-paring chloroquine and dofetilide are consistent with data ob-tained in the model of isolated canine Purkinje fibers (8) to predictproarrhythmic risk, which also showed chloroquine to be likely tobe associated with some arrhythmias.
Our results give a consistent message. DHA has little cardiactoxicity, as indicated in previous reports (49). PQP, which causesQT prolongation clinically but which has not been associated withTdP, demonstrated a blockage of the hERG channel which is notmediated by trafficking. In the rabbit ventricular wedge modelventricular preparations, PQP shows a low arrhythmogenic po-tential whether used alone or in combination with DHA.
In conclusion, it appears that DHA-PQP can be considered avaluable antimalarial therapeutic option from an in vitro cardiacsafety point of view, considering that (i) the malaria parasite isresistant to the combination of artemether and lumefantrine, witha �10% failure rate reported in Cambodia and resistance alsoreported in Ghana and Burkina-Faso (52), (ii) DHA combinedwith PQP has never been reported to induce sudden death inmalaria patients (57), and (iii) PQP is second only to chloroquine,in terms of human exposure, as treatment for malaria (37). Clin-ical pharmacovigilance data are needed to confirm this view.
ACKNOWLEDGMENTS
All contributors received financial support from Medicine for MalariaVenture to perform the present experiments.
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