Evaluation of cardiac liability of drugs by two in vitro functional assays

Shimin Wang, Teddy Lin, Karen Bernards, Yulia Ovechkina, Christine O’Day and Dan Small

MDS Pharma Services- Bothell, WA, USA

Drug induced cardiotoxic effects including delayed cardiac repolarization may induce arrhythmias

such as Torsades of Points or even sudden death. It is a challenge to detect these side effects of

compounds at the early stage of drug development in the pharmaceutical industry. To evaluate a

drug's potential to delay cardiac repolarization, two in vitro functional assays are widely employed: (1)

study of the effects of compounds on hERG K+ channel and (2) measurement of the action potential

duration in cardiac tissue. The hERG K+ channel assay is predominantly employed because most

drugs that reduce hERG K+ current delay cardiac repolarization. To test the cardiac liability of drugs,

these two assays were validated and their results were compared in this study. Eight known hERG K+

channel blockers were tested on HEK-293 cells expressing hERG K+ channel using the automated

voltage clamp technique. The calculated IC50 values of the testing compounds using the hERG K+

channel assay were comparable to those using the manual and automated patch clamp techniques

reported in literature . The effects of three of the eight known hERG K+ channel blockers on rabbit

Purkinje fiber action potential were also tested using the current clamp technique. D, L- Sotalol and

Dofetilide prolonged 50% and 90% action potential duration in a concentration dependent manner.

Quinidine also prolonged 90% action potential duration in a concentration dependent manner but a 10

µM concentration of Quinidine reduced the resting potential, slowed the maximum rate of rise of

action potential upstroke, decreased the amplitude of action potential and had no effect on 50% action

potential duration most likely because of its multiple channel inhibitory effects. These results match

those reported in literature. In general, the functional hERG K+ channel assay is a sensitive way to

detect cardiotoxic effect and it is cost effective and high throughput, while the functional action

potential assay provides relatively direct proarrhythmic information on drugs.

INTRODUCTION

hERG (human ether-a go-go-related gene) encodes human rapidly activating delayed rectifier potassium (K+) current (IKr) that is an essential component contributing to the repolarization of the cardiac myocyte action potential[1]. It is widely recognized that drug-related inhibition of hERG K+ channel results in a long QT syndrome that may trigger life-threatening arrhythmias such as Torsades de Pointes (TdPs) and even sudden death[2].

Since the last decade, many non-antiarrhythmic drugs have been withdrawn from the market mainly because of their inhibitory effect on the hERG K+ channel. Consequently, the S7B guideline issued by the International Conference on Harmonization (ICH) in 2002 recommends an in vitro evaluation of the effects of all pharmaceutical compounds that are targeted for human use on the hERG K+ channel (www.ich.org).

Electrophysiological techniques including voltage- and current- clamp techniques have provided the opportunity for translating key clinical liabilities into in vitro assays. Recent advances in the automated patch clamp technique enable broad screening of test compound effects on ion channels.

Using two different methods we studied (1) the inhibitory effects of eight known hERG K+

channel blockers on hERG K+ channel using PatchXpress 7000A. (2) the effects of three known hERG K+ channel blockers on Purkinje fiber action potential using current- clamp technique. (3) the possible relationship between hERG K+ channel blockage and action potential duration prolongation.

MATERIALS AND METHODS

hERG K+ current recording:

Cell culture: a HEK-293 cell line stably expressing hERG K+ channel was obtained from Dr. January’s laboratory[3]. The cells were cultured and harvested to obtain a suspension of single cells in extracellular solution.

Solutions and reagents: Intracellular solution contains (mM): KCl 130, Na-ATP 5, EGTA 5, MgCl2 5, HEPES 10, (pH 7.2). Extracellular solution contains (mM): NaCl 137, KCl 4, CaCl2 1.8, MgCl2 1.0, HEPES 10, Glucose 11, (pH 7.4). Eight known hERG K+ channel blockers: Astemizole, Ketoconazole, Quinidine, Verapamil, Cisapride, Terfenadine, Dofetilide and D,L- Sotalol were tested in this study.

Electrophysiological recording: hERG K+ currents were recorded using PatchXpress 7000A, a system for automated patch clamp (Molecular Devices Co., Sunnyvale, CA) in a whole-cell configuration. See Figure 1 and 2 for the pulse protocols. To test the reproducibility of the data, 3 sets of testing data were generated on 3 different days for 6 individual compounds and 1 set of testing data was produced from 2 individual reagents. At least 3 cells were obtained for each data set. The experiments were performed at ambient temperature.

Data analysis: Within each cellular recording, the current responses to test compound addition were normalized to the vehicle control and the percent of control values were calculated: ([current response / control peak tail current] x 100%). Means and standard errors were calculated for each test group. IC50 values were calculated using nonlinear regression to fit data to the Dose-Response, One-Site Model where: f = A + ((B-A)/(1+((C/x)^D))). Curve-fitting and IC50 calculations were performed using MathIQ™ software (IDBS).

Purkinje fiber action potential recording:

Fiber dissection, solutions and reagents: male rabbits (body weight 1.5 to 2.5 kg) were used for the

experiments. Rabbit Purkinje fibers were dissected surgically from either ventricles in a dissection

Tyrode solution containing (in mM): NaCl 118; KCl 30; CaCl2 1.8; MgCl2 1.0; NaH2PO4 1.8; NaHCO3

25; glucose 55 bubbled with 95% of O2 + 5% of CO2 and heated to 36.5 ± 1oC. The dissected

fibers were moved to a perfusion bath and superfused with normal Tyrode solution containing (in

mM): NaCl 118; KC1 4; CaCl2 1.8; MgCl2 1.0; NaH2PO4 1.8; NaHCO3 25; glucose 11 ( pH 7.40 ~

7.45) bubbled with 95% of O2 + 5% of CO2 and heated to 36.5±1oC. The fibers were continuously

perfused for at least one hour before action potentials were recorded. The microelectrodes were

fabricated and filled with 3M KCl with a resistance of 5 - 45 MΩ. Quinidine, Dofetilide and D, L

Sotalol were used in these experiments.

Action potential recording: Action potentials were recorded in current clamp mode using a Multi-

Clamp 700B amplifier. The Purkinje fiber was triggered by a 2 ms pulse with 2 fold of stimulating

threshold at a frequency of 1Hz or 2Hz.

The perfusion scheme is shown as follows:

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

Figure 1. hERG K+ channel current-voltage relationships and steady- state activation. hERG

steady- state activation (B) and deactivation currents (F) induced by stimulating pulses (A) and (E).

(C) Mean I-V relationship, (D) Steady- state activation. Data fit with Boltzmann equation: f =

(1/(1+exp(-(V-V1/2)/k))) and (G) Deactivation I-V relationship.

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 Ketoconazole. (C) Time course of

drug inhibition of hERG K+ currents.

Figure 3. Inhibition of hERG K+ currents by Astemizole, Ketoconazole, Quinidine, Verapamil,

Cisapride, Terfenadine, Dofetilide and D, L Sotalol. IC50 values were calculated using a

nonlinear regression to fit data to the Dose-Response, One Site mode: f = [A+((B-

A)/(1+((C/x)^D)))].

100000-

810000C,P10-110L9-350L2-45B140-800B300-1000B1700-2000I0.9-26BManual

Patch Data

reported in

literature

76400-

421000M,N

11-15A,M150D9-85A-K239-630A-K745–1300 A-K1863–2680A-

K

4 – 26A-KPatchXpress

Data

reported in

literature

417000

±±±±2085 ψψψψ15 ±±±±0.14 ψψψψ154.3 ±±±± 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 IC50 ± SEM values derived from three sets of experiments. ψψψψ Mean IC50 ± 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. www.milipore.com

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.

604±62*

(167%)

361±32

(100%)

525.4±66.9*

(172%)

306.2±23.3

(100%)

APD90 (ms)

305±45*248±28222.0±8.9216.0±18.2APD50 (ms)

245±72*430±65302.4±14.3*460.4±33.0Vmax (V/s)

105±2*120±2119.2±1.8*125.6±1.9APA (mV)

-79±5-89±1-84.9±0.3*-88.0±1.4RP (mV)

Quinidine 10 uMControl (1 Hz)Quinidine 10 uMControl (1 Hz)Parameter

Data from Literature4 (n=11)Our Data (n=5)

Figure 4. Effects of Quinidine on Purkinje fiber action potential and comparison of our current

clamp results with those reported in literature.

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±±±±28.7APD50 (ms)

--401.9±±±±38.3455.0±±±±24.2Vmax (V/s)

119.3±±±±13.1124.5±±±±6.1122.5±±±±1.7125.8±±±±2.1APA (mV)

-87.4±±±±2.2-91.1±±±±1.4-86.6±±±±1.5-91.9±±±±1.2RP (mV)

D,L-Sotalol 30 uMControl (1 Hz)D,L-Sotalol 30 uMControl (1 Hz)Parameter

Data from Literature5 (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.

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±33487±41420.2±14.6440.5±38Vmax (V/s)

131±6125±5125.3±1.5127.8±2.7APA (mV)

---90.1±3.2-89.7±3.7RP (mV)

Dofetilide 10 nMControl (1 Hz)Dofetilide 10 nMControl (1 Hz)Parameter

Data from Literature4 (n=11)Our Data (n=5)

Figure 6. Effects of Dofetilide on Purkinje fiber action potential and comparison of our current

clamp results with those reported in literature.

CONCLUSIONS

Eight known hERG K+ channel inhibitors were tested in this study using the automated

PatchXpress platform. Day to day variation of IC50 values obtained from three independent

experiments for 6 individual reagents were below 3-fold suggesting that this assay is consistent

and generates highly reproducible results.

IC50 values derived from this study are similar to the published values using both automated

and manual patch clamp techniques suggesting that the data generated in this study are

accurate and reliable.

Using the current clamp technique, we tested the effects of three known hERG K+ channel

inhibitors on rabbit Purkinje fiber action potential. D,L- Sotalol and Dofetilide prolonged 50%

and 90% action potential durations in a concentration dependent manner. Quinidine at 10 µM

reduced action potential amplitude, decreased the resting potential, depressed Vmax and

prolonged 90% action potential duration, but there was no effect on 50% action potential

duration, probably due to its multiple channel inhibitory effect. Our observed effects are

consistent with those reported in literature showing that this assay is reliable and can provide

high quality data.

The drugs that inhibit hERG K+ channel may also prolong action potential duration and

potentially induce long QT syndrome.

REFERENCES

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inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel.

Cell 81: 299-307.

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of the activation and inactivation kinetics of HERG Expressed in Xenopus oocytes. Journal of

Physiology 502(1):45-60.

3. Zhou Z, Gong Q, Ye B, Fan Z, Makielski JC, Robertson GA and January CT (1998). Properties

of hERG channels stably expressed in HEK 293 cells studied at physiological temperature.

Biophysical Journal 74:230-241.

4. Lu HR, Marien R, Saels A and Clerck FD (2001) Species plays an important role in Drug-

induced prolongation of action potential duration and early after depolarizations in isolated

purkinje fibers. Journal of Cardiovascular Electrophysiology, 12(1): 93-102.

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induced QT- prolongation in dogs with reduced repolarization reserve. Journal of

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