hERG SOT Poster 2010

In vitro cellular method development for evaluation of cardiac toxicity

1. Evaluationofcardiacliabilityofdrugsbytwoinvitrofunctionalassays ShiminWang,TeddyLin,KarenBernards,YuliaOvechkina,ChristineO’DayandDanSmall
MDSPharmaServices& Bothell,WA,USA
Drug induced cardiotoxic effects including delayedcardiac repolarization may induce arrhythmias
suchasTorsadesofPointsorevensuddendeath.Itisachallengetodetectthesesideeffectsof
compounds at the early stage of drug development in the pharmaceutical industry. To evaluate a
drug'spotentialtodelaycardiacrepolarization,twoin vitrofunctionalassaysarewidelyemployed:(1)
studyoftheeffectsofcompoundsonhERG K
+
channeland(2)measurementoftheactionpotential
duration in cardiac tissue. The hERG K
+
channel assay is predominantly employed because most
drugsthatreducehERG K
+
current delaycardiacrepolarization.Totestthecardiacliabilityofdrugs,
thesetwoassayswerevalidatedandtheirresultswerecomparedinthisstudy.EightknownhERG K
+
channelblockersweretested on HEK&293 cells expressing hERG K
+
channel using the automated
voltage clamp technique. The calculated IC
50
values of the testing compounds using the hERG K
+
channelassaywerecomparabletothoseusingthemanualandautomatedpatchclamptechniques
reportedinliterature.Theeffectsofthreeoftheeightknown hERG K
+
channelblockersonrabbit
Purkinjefiberactionpotentialwerealsotestedusingthecurrentclamptechnique.D,L& Sotalol and
Dofetilide prolonged50% and 90% action potential duration in a concentration dependent manner.
Quinidinealsoprolonged90%actionpotentialdurationinaconcentrationdependentmannerbuta10
@M concentration of Quinidine reduced the restingpotential, slowed the maximum rate of rise of
actionpotentialupstroke,decreasedtheamplitudeofactionpotentialandhadnoeffecton50%action
potentialdurationmostlikelybecauseofitsmultiplechannelinhibitoryeffects.Theseresultsmatch
thosereportedinliterature.Ingeneral,thefunctionalhERG K
+
channelassayisasensitivewayto
detect cardiotoxic effect and it is cost effective and high throughput, while the functional action
potentialassayprovidesrelativelydirectproarrhythmic informationondrugs.

2. INTRODUCTION
hERG (human ether&a go&go&related gene) encodes human rapidly activating delayed
rectifierpotassium(K
+
)current(IKr)thatisanessentialcomponentcontributingtothe
repolarizationofthecardiacmyocyte actionpotential
[1]
.Itiswidelyrecognizedthatdrug&
relatedinhibitionofhERG K
+
channelresultsinalongQTsyndromethatmaytrigger
life&threatening arrhythmias such as Torsades de Pointes (TdPs) and even sudden
death
[2]
.
Sincethelastdecade,manynon&antiarrhythmic drugshavebeenwithdrawnfromthe
marketmainlybecauseoftheirinhibitoryeffectonthehERG K
+
channel.Consequently,
theS7BguidelineissuedbytheInternationalConferenceonHarmonization(ICH)in2002
recommendsaninvitroevaluationoftheeffectsofallpharmaceuticalcompoundsthat
aretargetedforhumanuseonthehERG K
+
channel(
www.ich.org
).
Electrophysiologicaltechniquesincludingvoltage& andcurrent& clamptechniqueshave
providedtheopportunityfortranslatingkeyclinicalliabilitiesintoinvitroassays.Recent
advances in the automated patch clamp technique enable broad screening of test
compoundeffectsonionchannels.
Usingtwodifferentmethodswestudied(1)theinhibitoryeffectsofeightknownhERG K
+
channelblockersonhERG K
+
channelusingPatchXpress 7000A.(2)theeffectsofthree
knownhERG K
+
channelblockersonPurkinjefiberactionpotentialusingcurrent& clamp
technique.(3)thepossiblerelationshipbetweenhERG K
+
channelblockageandaction
potentialdurationprolongation.

3. MATERIALSANDMETHODS
hERG K
+
currentrecording:
Cellculture:aHEK&293celllinestablyexpressinghERG K
+
channelwasobtainedfromDr.January’s
laboratory
[3]
. The cells were cultured and harvested to obtain a suspension of single cells in
extracellularsolution.
Solutionsandreagents:Intracellularsolutioncontie1a.x-Mk/..ymg 20^o.8iV,sI.[o.:Ps,.[o.Mcmg
2
5,
HEPES10,(pH7.2).Extracellularsolutioncontains(mM):NaCl 137,KCl 4,CaCl
2
1.8,MgCl
2
1.0,HEPES
10, Glucose 11, (pH 7.4). Eight known hERG K
+
channel blockers: Astemizole, Ketoconazole,
Quinidine,Verapamil,Cisapride,Terfenadine,Dofetilide andD,L& Sotalol weretestedinthisstudy.
Electrophysiologicalrecording:hERG K
+
currentswererecordedusingPatchXpress 7000A,asystem
h R.iur -irpG.firEt.Egi-f.xM gpEugiR.CpFeEpa.m zo.lunnyvale,CA) inawhole&cellconfiguration.See
Figure1and2forthepulseprotocols.Totestthereproducibilityofthedata,3setsoftestingdata
weregeneratedon3differentdaysfor6individualcompoundsand1setoftestingdatawasproduced
from2individualreagents.Atleast3cellswereobtainedforeachdataset.Theexperimentswere
performedatambienttemperature.
Dataanalysis:Withineachcellularrecording,thecurrentresponsestotestcompoundadditionwere
normalized to the vehicle control and the percent of control values were calculated: ([current
response/controlpeaktailcurrent]x100%).Meansandstandarderrorswerecalculatedforeachtest
group.IC
50
valueswerecalculatedusingnonlinearregressiontofitdatatotheDose&Response,One&
Site Model where: f = A + ((B&A)/(1+((C/x)^D))). Curve&fitting and IC
50
calculations were performed
usingMathIQ™ software(IDBS).

4. Purkinjefiberactionpotentialrecording
:
Fiberdissection,solutionsandreagents: malerabbits(bodyweight1.5to2.5kg)wereusedforthe
experiments.RabbitPurkinjefibersweredissectedsurgicallyfromeitherventriclesinadissection
Tyrode solutioncontaining(inmM):NaCl 118;KCl 30;CaCl21.8;MgCl21.0;NaH2PO41.8;NaHCO3
25;glucose55bubbledwith95%ofO2+5%ofCO2andheatedto36.5± 1oC.Thedissected
fibersweremovedtoaperfusionbathandsuperfused withnormalTyrode solutioncontaining(in
mM):NaCl 118;KC14;CaCl21.8;MgCl21.0;NaH2PO4fXHP AnxBLR %OP sceVau4 ff ) Kx DXG< R 
7.45)bubbledwith95%ofO2+5%ofCO2andheatedto36.5±1oC.Thefiberswerecontinuously
perfused foratleastonehourbeforeactionpotentialswererecorded.Themicroelectrodeswere
fabricatedandfilledwith3MKCl witharesistanceof5& 45MS.Quinidine,Dofetilide andD,L
Sotalol wereusedintheseexperiments.
Actionpotentialrecording:Actionpotentialswereh4Vahl4l ri Vehh4it Vcn5K 5al4 euris n Mectrp
Bcn5K D<<, n5Kcr3r4hX 604 Deh9ri=4 3r24h 1nu thrss4redbya2ms pulsewith2foldofstimulating
thresholdatafrequencyof1Hzor2Hz.
Theperfusionschemeisshownasfollows:
Concentration 3
Vehicle
Concentration 2
Control
Washout
Concentration 1
t=0
t=15 min
t=25 min
t=35-65 min
t=45-75 min
t=55-85 min

5. Figure 1. hERG K
+
channel current-voltage relationships and steady- st ate activation. hERG
steady- state activation (B) and deactivation curren ts (F) induced by stimulating pulses (A) and (E). 
(C) Mean I-V relationship, (D) Steady- state activat ion. Data fit with Boltzmann equation: f = 
(1/(1+exp(-(V-V
1/2
)/k))) and (G) Deactivation I-V relationship. 

6. Figure 2. The development of steady- state block of  hERG K
+
currents by Ketoconazole. (A) Pulse 
protocol. (B) hERG K
+
currents inhibited by different concentrations of K etoconazole. (C) Time course of 
drug inhibition of hERG K
+
currents.

7. Figure 3. Inhibition of hERG K
+
currents by Astemizole, Ketoconazole, Quinidine, Ve rapamil, 
Cisapride, Terfenadine, Dofetilide and D, L Sotalol.  IC
50
values were calculated using a 
nonlinear regression to fit data to the Dose-Respon se, One Site mode: f = [A+((B-
A)/(1+((C/x)^D)))].

8. 100000-
810000
C,P
10-110
L
9-350
L
2-45
B
140-800
B
300-1000
B
1700-2000
I
0.9-26
B
Manual 
Patch Data 
reported in 
literature
76400-
421000
M,N
11-15
A,M
150
D
9-85
A-K
239-630
A-K
745–1300 
A-K
1863–2680
A-
K
4 – 26
A-K
PatchXpress
Data 
reported in 
literature
417000 
±±±±2085 
ψψψψ
15 ±±±±0.14 
ψψψψ
154.3±±±±13 * 12 ±±±±1 * 231 ±±±±32 * 1071 ±±±±89 * 2439 ±±±±206 * 20 ±±±±4 * MDS 
PatchXpress
Data
D, L-Sotalol
(nM)
Dofetilide
(nM)
Terfenadine
(nM)
Cisapride
(nM)
Verapamil
(nM)
Quinidine
(nM)
Ketoconazol
e
(nM)
Astemizole
(nM)
* Mean IC
50
±SEM values derived from three sets of experiments.  ψψψψMean IC
50
±SEM values derived from one set of experiments (n = 3).
A. J Biomolecular Screen 2005 10 (2):168-181.
B. Assay and Drug Development Technologies 2008 6(2):235-241.
C. 
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D-E. Automated Electrophysiology User Meeting. Molecular Devices, 2008.
F. Eur. Biophys. J 2009 38:273-278
G. J Pharmacol Toxicol Methods 2009 60(1):39-44.
H. Automated Electrophysiology User Meeting. Molecular Devices, 2005.
I. Br. J Pharmacol 2008 153:439-447.
J. Circulation 2002 105 :2830-2835.
K. J Pharmacol Experiment Therapeu 2000 292(1) :261-264.
L. Cardiovas. Res. 2004 (62):9-33.
M. J Pharmacol Toxicol Methods 2009 60(2):223-228.
N. Automated Electrophysiology User Meeting. Molecular Devices, 2006.
P. J Pharmacol Toxicol Methods 2004 50:93-101.
Table 1. Comparison of our PatchXpress results with  those reported in literature.

9. 604±62*
(167%)
361±32
(100%)
525.4±66.9*
(172%)
306.2±23.3
(100%)
APD90 (ms)
305±45* 248±28 222.0±8.9 216.0±18.2 APD50 (ms)
245±72* 430±65 302.4±14.3* 460.4±33.0 Vmax (V/s)
105±2* 120±2 119.2±1.8* 125.6±1.9 APA (mV)
-79±5 -89±1 -84.9±0.3* -88.0±1.4 RP (mV) 
Quinidine 10 uM Control (1 Hz) Quinidine 10 uM Control (1 Hz) Parameter
Data from Literature4 
(n=11) Our Data (n=5)
Figure 4. Effects of Quinidine on Purkinje fiber ac tion potential and comparison of our current 
clamp results with those reported in literature.

10. 735.7±±±±125.5*
(222%)
331.8±±±±34.6
(100%)
666.9±±±±60.7*
(190%)
349.0±±±±46.7
(100%)
APD90 (ms)
- - 502.4±±±±33.5* 276.8±±±±28.7 APD50 (ms)
- - 401.9±±±±38.3 455.0±±±±24.2 Vmax (V/s)
119.3±±±±13.1 124.5±±±±6.1 122.5±±±±1.7 125.8±±±±2.1 APA (mV)
-87.4±±±±2.2 -91.1±±±±1.4 -86.6±±±±1.5 -91.9±±±±1.2 RP (mV)
D,L-Sotalol 30 uM Control (1 Hz) D,L-Sotalol 30 uM Control (1 Hz) Parameter
Data from Literature
5
(n=6) Our Data (n=3)
Figure 5. Effects of D, L Sotalol on Purkinje fiber  action potential and comparison of our current 
clamp results with those reported in literature.

11. 444±43*
(167%)
266±14
(100%)
582.8±105.7*
(159%)
367.4±37
(100%)
APD90 (ms)
310±30*
(156%)
198±37
(100%)
420.5±100.9*
(162%)
259.7±43.9
(100%)
APD50 (ms)
511±33 487±41 420.2±14.6 440.5±38 Vmax (V/s)
131±6 125±5 125.3±1.5 127.8±2.7 APA (mV)
& & &90.1±3.2 &89.7±3.7 RP (mV) 
Dofetilide 10 nM Control (1 Hz) Dofetilide 10 nM Control (1 Hz) Parameter
Data from Literature
4
(n=11) Our Data (n=5)
Figure 6. Effects of Dofetilide on Purkinje fiber ac tion potential and comparison of our current 
clamp results with those reported in literature.

12. CONCLUSIONS
Eight known hERG K
+
channel inhibitors were tested in this study usingthe automated
PatchXpress platform. Day to day variation of IC
50
values obtained from three independent
experimentsfor6individualreagentswerebelow3&foldsuggestingthatthisassayisconsistent
andgenerateshighlyreproducibleresults.
IC
50
valuesderivedfromthisstudyaresimilartothepublishedvaluesusingbothautomated
and manual patch clamp techniques suggesting that the data generated in this study are
accurateandreliable.
Using the current clamp technique, we tested the effects of three known hERG K
+
channel
inhibitorsonrabbitPurkinjefiberactionpotential.D,L& Sotalol andDofetilide prolonged50%
and90%actionpotentialdurationsinaconcentrationdependentmanner.Quinidineat10@M
reduced action potential amplitude, decreased the resting potential, depressed Vmax and
prolonged 90% action potential duration, but therewas no effect on 50% action potential
duration, probably due to its multiple channel inhibitory effect. Our observed effects are
consistentwiththosereportedinliteratureshowingthatthisassayisreliableandcanprovide
highqualitydata.
The drugs that inhibit hERG K
+
channel may also prolong action potential durationand
potentiallyinducelongQTsyndrome.

13. REFERENCES
1.Sanguinetti MC.,JiangC.,CurranMEandKeatingMT(1995).Amechanisticlinkbetweenan
inheritedandanacquiredcardiacarrhythmia:HERGencodestheIKr potassiumchannel.
Cell81:299&307.
2.WangS.,LiuS.,MoralesM.,Strauss,HCandRasmusson RL(1997).Aquantitativeanalysis
oftheactivationandinactivationkineticsofHERGExpressedin Xenopus oocytes.Journalof
Physiology502(1):45&60.
3.ZhouZ,GongQ,YeB,FanZ,Makielski JC,RobertsonGAandJanuaryCT(1998).Properties
ofhERG channelsstablyexpressedinHEK293cellsstudiedatphysiologicaltemperature.
BiophysicalJournal74:230&241.
4.LuHR,Marien R,Saels AandClerck FD(2001)SpeciesplaysanimportantroleinDrug&
inducedprolongationofactionpotentialdurationandearlyafterdepolarizations inisolated
purkinje fibers.JournalofCardiovascularElectrophysiology,12(1):93&102.
5.Vormberge T,HoffmannMandHimmel H(2006)Safetypharmacologyassessmentofdrug&
inducedQT& prolongationindogswithreducedrepolarizationreserve.Journalof
PharmacologicalandToxicologicalMethods54:130&140.