Usefulness of combining multi-ion-channel assays with the Langendorff assay in an assessment of proarrhythmic risks

Mao YAMAGUCHI1, Koji NAKANO1, Satomi TOMIZAWA1, Rie URA1, Taku IZUMI1, Hironori OHSHIRO1, Hiroshi MATSUKAWA1, Akihiro KANNO1

1Drug Safety Testing Center Co., Ltd., Higashimatsuyama-shi, Saitama, Japan

Drug TdP Risk** Dofetilide

Ondansetron Moxifloxacin Dolasetron

Quinine Levocetirizine none

2017 Safety Pharmacology Society (SPS) Annual Meeting, September 2017, Berlin, Germany

Precise evaluation of cardiac safety in an early stage is essential for developing novel drugs. In particular, it is important to predict a possible risk for proarrhythmia in safety pharmacology testing. Prolongation of the QT interval, which is caused by blockade of the hERG channel, can lead to a life-threatening arrhythmia known as torsades de pointes (TdP). Although risk predictions by in silico model have been promoted, the models seem to have room for further improvement. Recently, the QT prolongation associated with blockade of other ion channels has also been discussed. So, we attempted to predict the proarrhythmic risks more accurately by combining ion-channel assays with the Langendorff assay. In this study, we tested 6 drugs, which had been evaluated in clinical QT prolongation assessments by IQ-CSRC*, using the manual patch-clamp technique and the Langendorff system.

* : Innovation and Quality in Pharmaceutical Development (IQ) and the Cardiac Safety Research Consortium (CSRC)

Materials & Methods

Cell Line Supplier Current

HEK-hNaV1.5 SB Drug Discovery INa,P, INa,L***

CHO-hCaV1.2/β2/α2δ1 ChanTest ICa

CHO-hKV4.3 B'SYS Ito

CHO-hKV1.5 CytoBioscience IKur

HEK-hERG University of Wisconsin-Madison IKr

CHO-hKVLQT1/mink CytoBioscience IKs

CHO-hKir2.1 B'SYS IK1

Drugs : 6 drugs which were used in the IQ-CSRC study

** : TdP risk categories by CredibleMeds (https://www.crediblemeds.org/)

: Known Risk of TdP : Possible Risk of TdP : Conditional Risk of TdP

Cell Lines : 7 stable cell lines Patch Clamp―Whole cell mode

Langendorff assay Animals : guinea pig Measurement parameters :

PR, QRS, RR, QT, HR, etc.

QTc and J-Tpeakc : Fridericia’s formula QTc or J-Tpeakc = (QT or J-Tpeak ) / RR1/3

These results revealed that the proarrhythmic risks are highly predictable by combining ECG analysis in the Langendorff system with multi-ion-channel profiling. The present study provided an insight into usefulness of combining multi-ion-channel assay with Langendorff assay for an assessment of proarrhythmic risks. We concluded that this combination assays will greatly contribute to the drug discoveries and developments.

Introduction

Results

*** : Late Na+ current (INa,L) was evoked by 50 μmol/L-veratridine.

If these channel are suppressed…

INa,L J-Tpeakc ↓

J-Tpeakc ↑ Tpeak-end ↑

QRS ↑

: QTc with 95% CI : J-Tpeakc with 95% CI : Tpeak-Tend with 95% CI

A

B INa,L

A : Johannesen et al., Clin Pharmacol Ther. 96(5):549-58 (2014) B : Gary Gintant et al., Nat Rev Drug Discov. 15, 457–471 (2016)

8.3 1.3 0

20

40

60

80

100

Dofetilide (100 μmol/L)

% b

lock

Concentration (nmol/L)

INa,P

ICa

11.7 24.0

5.7 0

20

40

60

80

100

Ondansetron (30 μmol/L)

% b

lock

INa,P

ICa INa,L

Concentration (μmol/L)

-20

0

20

40

60

80

100

0 10 20 30

IKr IC50 : 46.2 nmol/L

% b

lock

Concentration (nmol/L) Concentration (nmol/L)

Δ m

s 9

5% C

I

% ch

ange

9

5% C

I

Concentration (μmol/L) Concentration (μmol/L)

Dofetilide Ondansetron IKr inhibition : Strong ― IKr >>> INa, ICa

-20

0

20

40

60

80

100

0 10 20 30-101030507090

110130

1 3 10 30

% ch

ange

Dolasetron

-20

0

20

40

60

80

100

0 10 20 30

75.4

14.9 11.1 -1.0

-20

0

20

40

60

80

100

Dolasetron (30 μmol/L)

% b

lock

IKr IC50 : 9.2 μmol/L INa,P IC50 : 261.7 μmol/L

INa,P

ICa INa,L

IKr

Δ m

s 9

5% C

I

% ch

ange

9

5% C

I

Concentration (μmol/L) Concentration (μmol/L)

-20

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Levocetirizine

82.9

34.6 27.8 44.4

22.2 20.2 7.2 -1.5

-20

0

20

40

60

80

100

Levocetirizine (500 μmol/L)

% b

lock

48.0

7.2 5.3 6.6

-20

0

20

40

60

80

100

Levocetirizine (100 μmol/L)

% b

lock

-20

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

IKr IC50 : 124.6 μmol/L %

blo

ck

Concentration (μmol/L)

% ch

ange

9

5% C

I

Δ m

s 9

5% C

I

Concentration (μmol/L) Concentration (μmol/L)

IKr INa,P INa,L ICa

Ito IKur IKs IK1

IKr inhibition : Moderate ― IKr > ICa > INa Moxifloxacin

-20

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

31.6

7.6 14.6 24.4

0

20

40

60

80

100

Moxifloxacin (100 μmol/L)

% b

lock

-20

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

% ch

ange

9

5% C

I

Δ m

s 9

5% C

I

Concentration (μmol/L) Concentration (μmol/L)

INa,P

ICa INa,L

IKr

% b

lock

Concentration (μmol/L)

IC50, μmol/L

IKr 193.2 INa,P 2220.1 ICa 444.7

Quinine

-20

0

20

40

60

80

100

0 10 20 30

83.5

36.4 34.2 24.4

60.8 58.1

23.4

5.2 0

20

40

60

80

100

Quinine (30 μmol/L)

% b

lock

0

20

40

60

80

100

1 3 10 30

% ch

ange

-20

0

20

40

60

80

100

0 10 20 30

Δ m

s 9

5% C

I

% ch

ange

9

5% C

I

Concentration (μmol/L)

Concentration (μmol/L)

Concentration (μmol/L) Concentration (μmol/L)

: QRS duration : PR interval

IC50, μmol/L IKr 5.9

INa,P 46.1ICa 75.2Ito 19.6 IKur 19.7 IKs 162.8

Concentration (μmol/L)

IKr inhibition : Strong + Multichannel blocking

Conclusion

Strong IKr blocking

QTc ↑

J-Tpeakc ↑

Tpeak-end ↑↑

20 40 %

QRS ↑↑ PR ↑↑

Strong IKr blocking +

Multichannel blocking

QTc ↑

J-Tpeakc ↑

Tpeak-end ↑↑

10 20 %

QRS ↑↑ PR ↑↑

In the case of Levocetirizine… IKr : block

QTc ↑ J-Tpeakc↑ Tpeak-end ↑

80 %

60 %

IKr INa,P INa,L ICa

Ito IKur IKs IK1

% b

lock

IKr IC50 : 1.5 μmol/L

% b

lock

Results

Moderate IKr blocking

+ ICa, INa,P blocking

QTc ↑ J-Tpeakc ↑ Tpeak-end ↑

20 %

ICaL

IKr

INa,P ICa

% b

lock

Concentration (μmol/L)

IKr

INa,P

Ito1

INa

IK1

IKs

IKr

IKur

: QTc with 95% CI : J-Tpeakc with 95% CI : Tpeak-Tend with 95% CI

: QTc with 95% CI : J-Tpeakc with 95% CI : Tpeak-Tend with 95% CI

IKs

Ito IKur

x

IKr INa,P ICa

Concentration (μmol/L)

g

-20

0

20

40

60

80

100

0 10 20 30

Δ m

s 9

5% C

I

-20

0

20

40

60

80

100

0 10 20 30%

chan

ge

95%

CI

: QRS duration : PR interval