verification of cipa recommended voltage protocols in...

-400

0

400

800

1200

1600

2000

2400

2800

3200

3600

4000

0 10 20 30 40 50 60

Yoshimi KATAYAMA1),2), ○ Hiroshi MATSUKAWA1),3), Tomokazu KANEHISA1),4), Ayane ABE1),5), Takashi YOSHINAGA1),6), Keiichi ASAKURA1),7), Kimihito YOSHIKAWA1),8), Kazuya TSURUDOME1),9), Tadaaki TSUKAMOTO1),2)

1) iSmart, Japanese Safety Pharmacology Society, 2) Pharmaceutical Research Department, Biological Research Laboratories, Nissan ChemicalCorporation, 3) Higashimatsuyama Laboratories, Drug Safety Testing Center Co., Ltd., 4) Central Pharmaceutical Research Institute, Japan Tobacco Inc., 5)Drug Safety Evaluation, Research Laboratory for Development, SHIONOGI & CO., LTD., 6) Global Cardiovascular Assessment, Eisai Co., Ltd., 7)Pharmacokinetics and Safety Assessment Dept., Discovery Research Labs, NIPPON SHINYAKU CO., LTD., 8) Nonclinical Research Center, DrugDevelopment Service Segment, LSI Medience Corporation, 9) Sophion Bioscience K.K.

Verification of CiPA Recommended Voltage Protocols in Patch-Clamp Assay for hERG, Cav1.2 and Late Nav1.5 Currents

As revising an ICH regulatory guideline, S7B, for the Non-clinical Evaluationof the Potential for Delayed Ventricular Repolarization (QT IntervalProlongation by Human Pharmaceuticals) was suggested, Comprehensive Invitro Proarrhythmia Assay (CiPA) activities are being implemented. Thesuggested S7B revision includes an assay of cardiac ion channels other thanhERG, and CiPA-recommended voltage protocols for patch clamping wasrecently announced. We, Group 5 of the JSPS iSmart (investigation of in silico/ in vitro model for arrhythmogenic risk prediction) play a role of wet-datacollection. This time we have investigated the applicability of those protocols.

Introduction

Materials & Methods

Results & Discussion

Conclusions

Results of Cav1.2 Assay in Manual Patch-clamp

Comparison with CiPA Data Set

Results of hERG Dynamic Protocol

I-t Plot: Accumulative Application of Verapamil

Effect of Verapamil on hERG Current

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 10 20 30 40 50 60

Pulse No.

Am

plit

ud

e(p

A)

3 µmol/L

a

b c

a: control

b: 1st

c: 10th

Effect of Cisapride on hERG Current

Effect of E-4031 on hERG Current

b c

-100

0

100

200

300

400

500

600

700

800

900

1000

0 10 20 30 40 50 60

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 910

No

rm. (

%)

0

20

40

60

80

100

0 5000 10000

Time (msec)

A, I-t Plot B, Current Waveforms

C, Current Normalization (vs control)

2000 ms

500 pA

30 nmol/L

Am

plit

ud

e(p

A)

Pulse No.


C, Current Normalization (vs control)

2000 ms

1000 pA

0

20

40

60

80

100

0 5000 10000

No

rm. (

%)

Time (msec)


C, Current Normalization(vs control)

a

b

c

Am

plit

ud

e(p

A)

a

0

20

40

60

80

100

0 5000 10000Pulse No.Time (msec)

No

rm. (

%)

2000 ms

400 pA

Concentration-dependent Progressive Block

1 µmol/L

Comparison with CiPA Data Set3 μmol/L Ref 30 nmol/L Ref

200 msec 200 msec

IC50 (µmol/L) Hill n IC50 (µmol/L) Hill n

Bepridil 0.26 1.9 6-7 0.28 1.3 6Dofetilide 0.015 1.5 6-8 0.011 1.8 6Astemizole 0.026 1.4 6-9 0.026 1.3 6Chlorpromazine 1.7 2.0 6-7 0.85 1.6 6Cisapride 0.078 1.6 6-10 0.061 1.3 6Ranolazine 9.0 0.92 6 9.0 0.91 6Verapamil 0.90 2.1 7-10 0.63 1.2 6

Flecainide 1.8 1.0 7-8 1.2 0.98 6

DrugSite A Site B

IC50 (µmol/L) Hill IC50 (µmol/L) Hill

Bepridil 0.26 1.4 0.15 0.93Dofetilide 0.013 1.4 0.0015 0.63Astemizole 0.023 5.4 0.010 0.54Chlorpromazine 0.85 1.7 1.12 0.90Cisapride 0.071 1.9 0.012 1.3Ranolazine 3.4 1.1 6.5 0.84Verapamil 0.17 1.3 0.50 1.1

Flecainide －－－－

Li et al. 2018 Crumb et al. 2016Drug



Bepridil 0.34 1.9 1.8 1.4

Astemizole 0.60 3.1 10 2.3

Chlorpromazine 0.67 1.8 4.6 0.94

Ranolazine 6.0 0.99 7.9 0.95

Verapamil 0.98 1.2 24 2.0

Flecainide －－－－

Li et al. 2018 Crumb et al. 2016Drug

Results of hERG Assay on QPatch with IC50 Only Protocol

I-t Plot

The compound was injected 8 times (single application) or 4 times (accumulative application) at every 20 pulses.

Current Traces

Results of Late Nav1.5 Assay on QPatch

Effect of Ranolazine on Late Nav1.5 Current

Current Traces


A

BA

B

20 msec

1000 pA

20 msec

1000 pA

A: −15 mV Step B: Ramp down

150 nmol/L ATX-II

10 μmol/L Ref100 μmol/L

Inter-facility Difference in Enhancement by ATX-II

The results show that enhancement of late Nav1.5 current by ATX-II differs between facilities and that a distinct combination of intra- or extra-cellular solutions (see below) sufficiently induced late Na current in an identical platform (site C) where late Na current could be hardly detected with CiPA conditions in the presence of ATX-II.Note: Solutions (in mmol/L): EC, NaCl 145, KCl 4, CaCl2 2, MgCl2 1, glucose 10, HEPES 10, pH 7.4 with NaOH; IC, NaCl 10, CsF 135, EGTA 1, HEPES 10, pH 7.3 with CsOH.

Site A(CiPA sol.)

Site C(CiPA sol.)

Site C(Original sol.)

Site E(CiPA sol.)

Time (msec)

0 100005000

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 910

Verapamil

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 910

1st episode 10th episode

Time (msec)

Frac

tio

nal

blo

ck (

%)

0 0 50005000 10000 100000

20

40

60

80

100

0 1 2 3 4 5 6 7 8 910

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 910

Cisapride

1st episode 10th episode

Time (msec)

0 05000 500010000 10000

Frac

tio

nal

blo

ck (

%)

Frac

tio

nal

blo

ck (

%)

(n=4 each)

a: control

b: 1st

c: 10th

a: control

b: 1st

c: 10th

1st10th

1st10th

1st10th

3 µmol/L

1 µmol/L

0.3 µmol/L

0.1 µmol/L

30 nmol/L

10 nmol/L

3 nmol/L

1 nmol/L

n=3 or 4 each

(n=4 each)

-1000

-900

-800

-700

-600

-500

-400

-300

-200

-100

0

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1 µmol/LNife

0.1 µmol/L

a c

d

b

Time (min)

Am

plit

ud

e (p

A)

Verapamil

Suppression rate (%) : 80.3± 4.8 (for initial peak), 91.5± 5.3 (for steady state)

IC50 (µmol/L) Hill n IC50 (µmol/L) Hill n

Bepridil 0.031 1.3 6 0.10 1.6 7-8Dofetilide 0.0095 1.2 6 0.014 1.8 6-8Astemizole 0.0042 1.2 6 0.0066 1.00 6-9Chlorpromazine 0.17 1.2 6 0.50 1.3 6-8Cisapride 0.0080 0.93 6 0.019 0.93 6-11Ranolazine 3.5 0.89 6 5.2 0.89 7-9Verapamil 0.13 1.1 6 0.35 0.90 6-8

Flecainide 0.44 0.82 6 0.59 0.90 6-8

DrugSite C Site D

200 msec

30 nmol/L Ref

Ranolazine Cisapride Dofetilide

30 μmol/L

a

c

d

ba

c

b

a

cb

a

cb

a

c

b

a

c d

b

2000 pA 1000 pA 1000 pA 500 pA

ATX-II (−)

ATX-II (+)

ATX-II (−)

ATX-II (+)

ATX-II (−)

ATX-II (+)

ATX-II (−)

ATX-II (+)

150 nmol/L ATX-II

10 μmol/L Ref100 μmol/L


Bepridil 1.9 1.5 1.2 0.5 3-5

Astemizole 1.7 1.8 1.1 1.3 3-6

Chlorpromazine 1.9 2.6 1.3 1.9 3-4

Ranolazine 52 1.7 29 1.7 7-8

Verapamil 14 1.4 6.3 1.3 5

Flecainide 1.5 1.4 1.3 1.0 3

Drug

Site A

−15 mV Step Ramp downn

Manual patch clamp and an automated patch-clamp system, QPatch, wereselected as the platforms. The manual system was used for hERG dynamicprotocol (physiological temperature) and Cav1.2 (room temperature);QPatch for hERG IC50 only protocol and late Nav1.5 (room temperature). Testcompounds were selected from each of the low-, intermediate-, and high-risk categories of the “training drugs” in the CiPA in-silico model. Flecainidewas also selected.

CiPA compounds used:

Cell lines: hERG-HEK293/CHO-K1 cells, Nav1.5-HEK293/CHO-K1/CHL cells, Cav1.2-CHO.

Patch-clamp conditions: identical to what the CiPA initiative announcedunless otherwise stated.

Flecainide

-80 mV

+40 mV, 500 msec

100 msec

1.2 V/sec

-90 mV, 100 msec

-80 mV

IC50 protocols

hERG

hERG dynamic model protocols

-80 mV

0 mV, 10000 msec

-90 mV, 100 msec

-80 mV-80 mV

-90 mV, 100 msec

-80 mV

Pulse No-pulse

-120 mV,200 msec

+40 mV, 200 msec

-15 mV,40 msec

100 msec

-95 mV-95 mV,50 msec

1.35 V/sec

Nav1.5 (Late Na)(every 10 sec)(every 5 sec)

Cav1.2

0 mV,40 msec

100 msec

1.1 V/sec

+30 mV, 200 msec

-80 mV

-90 mV, 100 msec

-80 mV

(every 5 sec)

(every 25 sec, 10 pulses) (every 25 sec, 10 pulses)

Alternatively apply

plus

TdP risk High Intermediate Low

Bepridil Astemizole Ranolazine

Dofetilide Chlorpromazine Verapamil

Cisapride

CiPA

drug

a

c

d

b

Analysis

Cav1.2 current waveforms Subtraction with nifedipine treatment

50 ms

200 pA

-10 mV Voltage step

AcknowledgementWe would like to express our gratitude to Sophion Bioscience K.K. for the courtesy ofQPlates and the solid technical support with respect to data acquisition and analyses onQPatch.

References• Recommended voltage protocols to study drug-cardiac ion channel interactions using

recombinant cell lines• Journal of Pharmacological and Toxicological Methods, Crunb etal., 2016• Circ Arrhythm Electrophysiol, Li et al., 2017• CLINICAL PHARMACOLOGY & THERAPEUTICS, Li et al., 2018

• Some of our data which disagree with those presented by CiPA arediscussed. Some problem and suggested modifications are pointed. Weplan to deliver these suggestions to CiPA.

• In hERG IC50 only protocol, the similar results were obtained amongdifferent facilities after optimizing the duration and number ofapplication on QPatch system.

• In late Nav1.5 current protocol, it was turned out that enhancement oflate Na current by ATX-II was not consistent among different facilitieswhen CiPA solutions were used and was dependent on the combinationof intra- and extra-cellular solutions, suggesting the possibility that theprotocol may still have some room for improvement.

• In Cav1.2 current protocol, the IC50 values of dofetilide and cisapridedeviated far from the results by CiPA.

• In a limited number of compounds from intermediate and low riskcategories, the IC50 values were almost similar to the results by CiPA.

a

c

b

Initial peak

2nd peak

Initial peak

2nd peak

nIC50

(µmol/L)Hill

IC50

(µmol/L)Hill

IC50

(µmol/L)Hill

Cisapride 1, 3, 10, 30* 3 or 4 NA NA 10.8* 1.11 10.1* 0.7

Ranolazine 1, 3, 10, 30 3 or 4 13.5 0.67 8.50 0.89 8.27 0.9

Verapamil 0.1, 0.3, 1, 3 4 2.48 0.60 0.369 0.85 0.288 1

Flecainide 0.3, 1, 3, 10 4 2.47 0.65 1.16 0.86

*: Cisapride concentrations are shown in nmol/L. NA: Not applicable.

Li et al. 2017, 2018

Drug

Test

concentration

(µmol/L)

Initial peak Steady state

n IC50 Hill IC50 Hill IC50 Hill IC50 Hill

Bepridil 0.1, 0.3, 1, 3 4 0.392 1.08 0.365 1.22 2.82 0.65 638 4.6

Dofetilide# 100 4 >> 100*1 ー >> 100*2 ー 44.5 3.6 2.30E+03 5.4

Astemizole# 0.03, 0.1, 0.3, 1 4 0.192 1.20 0.217 1.18 0.553 1.2 1.08 5.9

Chlorpromazine# 0.1, 0.3, 1, 3 4 0.741 1.59 0.728 1.98 8.32 0.85 6.35 2

Cisapride 0.3, 1, 3, 10 4 2.65 1.08 1.49 1.24 1.03E+03 4.8 4.05E+03 5.6

Ranolazine 30, 100, 300, 1000 4 514 0.99 324 0.92 900 3.9 6.54E+03 3.8

Verapamil 0.1, 0.3, 1, 3 4 0.387 0.98 0.509 1.02 0.204 1.1 11.2 0.8

Flecainide 3, 6, 30, 60 4 23.8 0.88 21.5 1.00

#: Each cell was exposed to single drug concentration.

Manual*3 HTSDrug

*1: 8.6% at 100 µmol/L . *2: 5.7% at 100 µmol/L . *3: Action potential protocol, Ba2+ as charge carrier, at physiological

temperature.

Crumb et al. 2016 Li et al. 2018

Test conc.

(µmol/L)

Initial peak 2nd peak

verification of cipa recommended voltage protocols in...

Documents