Human tissue cover 2014 Tissu… · CV T CV L Electrophysiology: Conduction Velocity Pharmacology: E-4031, Chromanol 293B and 4-AP A C E-4031 Figure 6 (IIHFW RI WKH ,.U LQKLELWRU(

Posters

0.5 Hz 1 Hz 2 Hz200250300350400450500550600

FPD

(ms)

1h 4h 8h50

60

70

80

90

100

110

FPD

Control

100

M1m

M WO0

20406080

100120140160 **

*

FPD

(%)

Control

2min

5min

10min

15min WO

020406080

100120140160

**** **

*

FPD

(%)

Control

2min

5min

10min

15min WO

50

75

100

125 ** **

FPD

(%)

Patrizia Camelliti1,2,3, Sara Abou Al-Saud2, Ryszard T. Smolenski4, Samha Al-Ayoubi2,Nicholas Banner2, Christopher Bowles2, Magdi Yacoub2 & Cesare Terracciano2

1School of Biosciences and Medicine, University of Surrey, UK2Harefield Heart Science Centre, Imperial College London, UK

3University of Oxford, UK 4Medical University of Gdansk, Poland [email protected]

MethodsCardiac Slice Preparation

Morphology

Metabolic Analysis Essay

Electrophysiology

Pharmacology

niversity of Gdanskniversity oof Gdansof G skk,k

audSS ddaudSS ddddd ,,,,, RyszarR rddyszarRyRR ardarrdd TTowlesowlesowo sssss222,,, MagdMagdMMM dddidig YaYaYaYaYYYand Medicine, Univand MMeedicineediced UnivUniUnand Medicinne, Univdicin nive Centree Centree,ee Centree, mperiamperiaImIm alalmperiaIm al CCCi it Gdf G kk

Introduction

human myocardium

human cardiac slice

Metabolism

Acknowledgements

Conclusions

Patriziaa CamellitC ti 21,22,33 araS a boA ouu AAAlA - TTTT molenskS kki44ii amhS haa AAAlA - youbAyA bbi- 22iiaudSS dd22d RyszarR rdd TaudSS dd22dd RyszarR rdd THuman Cardiac Slices: A Novel Tool for Drug Safety Screening

Figure 1 AB

C

ResultsCardiac Slice Morphology

A BC

Figure 2A B

C DC D

0

10

20

30

40

50

60

70

CV

(cm

/s)

Figure 4 A

B

C

C

CVTCVL

Electrophysiology: Conduction Velocity

Pharmacology: E-4031, Chromanol 293B and 4-AP

A C

E-4031

Figure 6 A BC

vs

A B C

A B

DB

A B

C D

Figure 3A

B

A B

Electrophysiology: Field Potential Duration

Chromanol 4-AP

D

vs D

Figure 5 A CA B A

C D

www.ucb.com

3

Why a Stem Cell Approach in Investigative Toxicology ? Currently, drug induced cardiac safety liabilities are typically assessed during the early stages of drug discovery by evaluating effects on human cardiac ion channels in vitro, cardiovascular function in vivo and histopathology.

The project aims to validate induced pluripotent stem cells (iPS) derived cardiomyocytes (CM) to:

- Assess pro-arrhythmia liability in a cellular model

- Provide a new tool for inotropy/chronotropy assessment

- Set-up a first in vitro integrated cardiotoxicity assay at UCB

From our hypothesis, such a model could complement the electrophysiological assessment done in vitro and help identifying other potential cardiac liabilities before in vivo studies.

We investigated and compared a well characterised mouse iPSCM model to human iPSCM that may be more relevant to clinical situation.

Comparison of mouse and human iPS cardiomyocytes models to detect cardiac safety liabilities

Aurore Colomar, Benjamin Kopp, Lucille Haen, Gaëlle Toussaint, Franck Atienzar, Annie Delaunois & Jean-Pierre Valentin

Non-Clinical Development – UCB BioPharma, Braine-l'Alleud, Belgium

Results

Conclusion-Perspective 5/7 compounds induced decrease in CI in h and m iPSCM.

6/7 compounds induced decrease in BR with increase in AC (3/7), decrease in AC (2/7) or different effects on AC in h and m iPSCM (1/7).

1/7 compound induced increase in BR and decrease in AC.

3/4 compounds induced effects in iPSCM that correlated with ex vivo or in vivo findings. However, other investigations are required to assess further the predictivity of the model.

Magnitudes of effects were higher on CI, BR and AC in hiPSCM compared to miPSCM. Moreover concentrations inducing effects on BR and AC were lower in hiPSCM vs miPSCM.

Control conditions2 Material & Methods

Parameters evaluated:

iPS derived Cardiomyocytes:

We compared the ability of hiPSCM and miPSCM to detect cardiotoxicity by evaluating the effects of 7 compounds on Cell Index (CI), Beating Rate (BR) and Amplitude of Contraction (AC) of iPS-CM using an impedance based measurements (RTCA cardio, ACEA, San Diego, USA).

Human (h) cardiomyocytes derived from induced Pluripotent Stem cell hiPS-CM (iCell, CDI, Madison, USA) were cultured in a 96 well e-cardio Plate at 20.000 cells/well during 11 days.

Mouse (m) iPS-CM (CorAt, Axiogenesis, Köln, Germany) were cultured at 40.000 cells/well during 3 days.

Both cell types were treated with 7 compounds discarded from development due to cardiac liabilities or suspected to affect cardiac activity (n 3 wells/concentration).

At the next medium change (24h for hiPS, and 16/18h for miPS) a 4h recovery period was allowed.

Principle of impedance-based recording:

Adapted from Yamanaka et al., Nature, 2010

y y

AdapteAdaptedd fromfrom YamanaYamanakaka et alet al Nat., Nature 2ure, 2010010

Differentiation in

Cardiomyocytes

- actinin labeled mouse Cardiomyocytes

H3R antagonist

W d th bilit f hiPSCM d iPSCM t d t t

Principle of impedance based recording

Xi et al., J. Laboratory Automation, 2011

Amplitude of Contraction &Beating Rate

BR

AC

Cell Index & Normalized Cell

Index

ucb34742

ucb106607

S1P3R antagonist

4 Barriers in adopting human tissue approaches RTCA cardio analysis has the potential advantage to detect effects on contraction and indirectly on viability and electrophysiology. However, it necessitates synchronized beating of iPSCM with sufficient amplitude to be detected by the system. This was a particular issue with the human iPSCM.

This issue included technical hurdle for cell provider to develop mature cells that generate recordable contraction and access from companies to such cells.

Long duration culture and variability of parameters are issues specific to hiPSCM that still need to be overcome.

3 Rs impact

hiPSCM Pre-treatment(n=96)

DMSO 0.1%Final read beforewashout (n=12)

% change DMSO vs pre-treatment

Cell Index 6.46±1.1 4.99±0.96 -22%

Beating Rate 34.29±6.44 25.26±15.27 -26%

Amplitude of Contraction

0.08±0.02 0.04±0.03 -48%

miPSCM Pre-treatment(n=958)

DMSO 0.1% at 12h post dose (n=96)

% change DMSO vs pre-treatment

Cell Index 11.68±1.5 12.02±1.7 +3%

Beating Rate 116.5±25.9 94.17±20.32 -19%

Amplitude of Contraction

0.33±0.11 0.38±0.12 +15%

% change vs DMSO

miPSCMCI

hiPSCMCI

miPSCMBR

hiPSCMBR

miSPCMAC

hiPSCMAC

miPSCMrecovery

hiPSCMrecovery

ucb106607-59%

30μM

-25 to -94%

10 & 30μM

-27%

30μM

-17 to -100%

1 to 30μM

-100%

30μM

-12 to -100%

1 to 30μM

AC (partly) & CI recoveredBR rebound

CI, BR, AC partly

No recovery at30μM

ucb34742-13 to -29%

10 & 30μM

-26 to -39%

10 & 30μM

-11 to -20%

3 to 30μM

-16 to -100%

1 to 30μM

-20 to -77%

10 & 30μM

-11 to -100%

1 to 30μM

CI (slightly), BR, AC recovered

CI, BR, AC slightlyrecovered

H3 antago +3%

30μM

-4.5%

30μM

-15%

30μM

-24 to -62%

10 & 30μM

+10 to +17%

3 to 30μM

-13 to -46%

10 & 30μM

AC recoveredBR rebound

AC (partly), BR, CI recovered

S1P3 -1%

30μM

-21%

30μM

+8 to +16%

10 & 30μM

+8 to 53%

1 & 10μM

-27%

30μM

-9 to -100%

10 & 30μM

BR recoveredNo recovery on AC at

30μM

CI, AC (partly) & BR recovered

Kinase inhibitor 1 -10 to -22%

10 & 30μM

-4 to 6%

15 & 30μM

-7%

at 30μM

-10%

at 30μM

+5%

10μM

+16 to 21%

15 to 30μMCI partly recovered

CI, BR partlyrecovered

No recovery on AC

Kinase inhibitor 2 -14%

at 30μM

-10%

at 30μM

-6 to -9%

at 10 & 30μM

-15 to -31%

7.5 to 30μM

+7%

10μM

+16 to 19.7%

3.75 to 30μM

CI partly recovered

Irregularity atwashout

CI, BR partlyrecovered

No recovery on AC

Kinase inhibitor 3-9 to -28%

3 to 30μM

-8 to -14%

7.5 to 30μM

-8%

at 10 & 30μM

-10 to -22%

7.5 to 30μM

+17%

10μM

+7.3 to 44%

0.47 to 30μM

CI partly recovered

Irregularity atwashout

Partly recovered

CI

The RTCA cardio analysis of iPSCM contractions is an in vitro model using a single biopsy of mature cell as basis. Per se, it has no impact on animals usage.

This model might help to identify cardiac liabilities at early stage of development and offers suitable alternative to replace ex vivo models such as Purkinje fibre and isolated atria.

Currently, the use of RTCA cardio/iPSCM could help to identify hazard on parameters requiring follow-up in in vivo studies and to triage compounds. It may reduce the animal use as it allows a better selection of appropriate compounds to be tested in vivo.

However, further investigations are required to investigate the potential of the model to detect chronotropy, inotropy and iPSCM dysfunction leading to structural cardiac toxicity that are usually detected in in vivo models.

ucb106607

ucb34742

H3 antago

S1P3 antago

SSRI stopped during phase I study due to severe cardiac adverse events (AE): cardiac atrial pauses and AV block II with junctional escape rhythm. ucb106607 has multi-ion channel blocker activity, however the mechanism of the AE is not fully understood

5LO-H1 antagonist stopped during preclinical development due to QTc prolongation in dog repeated-dose tox studies. ucb34742 is a weak hERG inhibitor (IC50= 21.8μM)

H3R antagonist stopped during preclinical development due to hERG liability (IC50= 8.6μM) translating into APD prolongation, Reverse Use Dependence and Triangulation in rabbit Purkinje Fibre assay at the expected therapeutic concentration

S1P3R selective antagonist suspected to induce increase in Beating Rate via its primary pharmacological activity

hiPSCM profiles of contraction

Comparison of drug-induced effects on CI, BR & AC on h & m iPS cardiomyocytes

In brown: effects on mouse iPSCM In bold: highest sensitivity of human vs mouse iPSCM

In red: effects on human iPSCM In italics: specific effects on mouse iPSCM

In hiPSCM, H3R antagonist reduced Beating Rate and Amplitude of Contraction in a concentration-dependent manner that became significant after 15 min (BR) and an hour (AC) of treatment and persisted through 24 hours.

In miPSCM, the compound induced opposite effect on Amplitude of Contraction and transient irregularity of beating (see table).

In hiPSCM, ucb106607 reduced dramatically Beating Rate and Amplitude of Contraction in a concentration-dependent manner that led to cessation of beating from 5 min to 14h after treatment at 10 and 30μM. The Cell Index decreased and was not rescued after washout likely due to a cytotoxic effect at 30μM.

In miPSCM, the compound induced similar effect but of lower magnitude than in hiPSCM (see table).

In hiPSCM, ucb34742 reduced Beating Rate and Amplitude of Contraction in a concentration-dependent manner that led to reversible cessation of beating at 10 & 30μM. The Cell Index decreased at 10 and 30μM but was partly rescued after washout.

In miPSCM, the compound induced similar effect but of lower magnitude than in hiPSCM (see table).

In hiPSCM, S1P3 antagonist induced concentration-dependent increase in Beating Rate followed by cessation of beating at 30μM.

In miPSCM, the compound induced increase in Beating Rate of lower magnitude than in hiPSCM (see table).

1

•

Hor

tig

on-V

inag

re,

MP

1;

Gh

ouri

, IA

1;

Cra

ig,

MA

2;

Bu

rton

, FL

1,2

;K

ette

nh

ofen

, R

3,

Sch

wen

gb

erg

,S3

,4,

and

Sm

ith

, G

L1,2

1 Insti

tute

of C

ardio

vasc

ular a

nd M

edica

l Scie

nces

, Univ

ersit

y of G

lasgo

w, Sc

otlan

d; 2 C

lyde B

ioscie

nces

, Univ

ersit

y of G

lasgo

w, Sc

otlan

d; 3 A

xioge

nesis

AG,

Colo

gne,

Germ

any;

4 Cell

s At W

ork,

Germ

any

Ap

plic

atio

n o

f op

tica

l mea

sure

men

ts o

f el

ectr

ical

act

ivit

y to

iPS

c-d

eriv

ed c

ard

iom

yocy

tes

as a

hig

h-t

hro

ug

hp

ut

pre

dic

tive

too

l for

pre

clin

ical

saf

ety

asse

ssm

ent

Intr

odu

ctio

nA m

ajor

con

cern

dur

ing

drug

dev

elop

men

t is

car

diac

tox

icity

. Th

e cl

assi

cal e

lect

roph

ysio

logi

cal t

echn

ique

s, a

imed

to

stud

y th

e ca

rdia

c ac

tion

pote

ntia

l (AP)

, su

ch a

s th

e m

anua

l pat

ch

clam

p an

d m

icro

elec

trod

e ar

ray,

are

low

thr

ough

put

and

tech

nica

lly d

eman

ding

. Th

eref

ore

the

deve

lopm

ent

of n

ew t

echn

olog

ies

to im

prov

e th

ese

limita

tions

is

impe

rativ

e. I

n th

is

wor

k w

e pr

esen

t th

e ut

ility

of

Cel

lOPT

IQ (

Cly

de B

iosc

ienc

es),

an

optic

al s

yste

m t

o de

tect

the

ele

ctrica

l ac

tivity

of

card

iac

cells

, in

con

junc

tion

with

ind

uced

plu

ripo

tent

ste

m c

ell

derive

d ca

rdio

myo

cyte

s (i

PSC-

CM

s) a

s an

alte

rnat

ive

to o

verc

ome

the

bott

lene

ck o

f lo

w t

hrou

ghpu

t an

d su

itabl

e tis

sue

cells

ass

ocia

ted

with

tra

ditio

nal c

ardi

ac A

P st

udie

s

Res

ult

sTh

e m

ost

rele

vant

res

ult

was

the

dem

onst

ratio

n of

hER

G r

eact

ivity

in

a m

urin

e de

rive

d iP

SC-

CM

(Cor

.At®

Cel

ls).

A d

ose

depe

nden

t pr

olon

gatio

n of

AP

dura

tion

(APD

) w

as c

ause

d by

the

wel

l-kn

own

hERG

bl

ocke

r E4

031,

whi

ch a

lso

indu

ced

EAD

s an

d ta

chya

rrhy

thm

ic e

ffec

ts a

t th

e hi

ghes

t co

ncen

trat

ions

em

ploy

ed (

30 a

nd 1

00 n

M r

espe

ctiv

ely)

. Th

e APD

at

90%

rep

olar

izat

ion

afte

r in

cuba

ting

the

cells

with

10n

M

E403

1 fo

r 30

min

is 7

07.8

5±99

.85

ms,

rep

rese

ntin

g a

237%

incr

ease

abo

ve b

asel

ine

(APD

90 =

289

.35±

1.65

ms)

.

hERG

rea

ctiv

ity h

as a

lso

been

sho

wn

in h

uman

iPSC-

CM

s (C

or.4

U®

). A

dos

e de

pend

ent

incr

ease

of

APD

90 is

sho

wn,

at

100

nM E

4031

APD

90 w

as 1

70 ±

2.6%

of

base

line

(APD

90 =

286

.9 ±

0.7

ms

and

487.

4 ±

8.7

ms,

bas

elin

e an

d tr

eatm

ent

resp

ectiv

ely)

. N

o ch

ange

(10

2 ±

1.8

% o

f ba

selin

e) w

as p

rese

nt w

hen

cells

are

tre

ated

with

veh

icle

.

Inhi

bitio

n of

L-t

ype

Ca2

+ c

hann

el b

y ni

fedi

pine

was

als

o te

sted

. Th

e APD

at

75%

rep

olar

izat

ion

was

86.

3 ±

5.1

% a

nd 7

7.2

± 3

.5%

of

base

line

whe

n th

e Cor

4U

cel

ls a

re t

reat

ed w

ith n

ifedi

pine

0.1

and

0.3

μM

resp

ectiv

ely.

No

chan

ges

wer

e ob

serv

ed a

t th

e sa

me

vehi

cle

conc

entr

atio

n (A

PD75

= 1

07 ±

4.4

%

and

104

± 4

% r

espe

ctiv

ely)

.

Met

hod

To c

arry

out

the

stu

dies

, iP

SC-

CM

s (A

xiog

enes

is)

plat

ed i

n 96

wel

l gl

ass

bott

om p

late

(20

,000

cel

ls/w

ell)

wer

e tr

ansi

ently

loa

ded

with

6 μ

M d

i-4-

AN

EPPS

and

the

car

diac

ele

ctrica

l ac

tivity

was

m

onito

red

from

spo

ntan

eous

ly b

eatin

g iP

SC-

card

iom

yocy

tes

at a

dat

a sa

mpl

ing

rate

of

10,0

00 H

z ov

er 3

0 se

cond

s tim

e w

indo

ws

empl

oyin

g th

e pl

atfo

rm C

ellO

PTIQ

, an

d th

e re

cord

s w

ere

subs

eque

ntly

ana

lyse

d of

f-lin

e us

ing

prop

riet

ary

soft

war

e (C

lyde

Bio

scie

nces

). T

he s

tatis

tical

ana

lysi

s w

as d

one

by u

npai

red

T-te

st (

*p<

0.05

, **

p<0.

005,

***

p<0.

0005

) C

ellO

PTIQ

exc

= 47

0nm

em

s1 <

585

nm

ems2

> 5

85nm

di-4

-AN

EPPS

(Vol

tage

sen

sitiv

e dy

e)

Ave

rage

d

iPSC

-CM

s

0.01

0.1

110

100

100

200

300

400

500

600

700

800

**

APD90, ms

[E40

31],

nM

Veh

icle

(wat

er)

E40

31

EADs

Ta

chy

0.01

0.1

110

100

0.6

0.8

1.0

1.2

EADs

Ta

chy

Beating Frecuency, Hz

[E40

31],

nM

Veh

icle

(E40

31)

E40

31

1 se

c1

sec

TREA

TMEN

TBA

SELI

NE

E403

1 30

nMVe

hicl

e (w

ater

)

1 se

c

Pre-

Vehi

cle

(wat

er)

1 se

c

Pre-

E403

1 30

nM

Ba

selin

eD

rug

200

ms

Ba

selin

eD

rug

200

ms

Ba

selin

eD

rug

200

ms

Ba

selin

eD

rug

200

ms

E403

1 10

0nM

E403

1 30

nME4

031

10nM

Vehi

cle

(wat

er)

Cycl

e Le

ngth

, sec

0.56 ±

0.01

0.58 ±

0.02

0.65 ±

0.0

0.75 ±

0.01

Dia

stol

ic p

erio

d,

sec

0.32 ±

0.01

0.31 ±

0.01

0.32 ±

0.00

30.3

1 ± 0.

0

APD

90, m

s24

0.3 ±

4.1

264.3

± 8.

032

9.5 ±

3.55

443.6

± 9.

9Fr

ider

icia

’s

Corr

ecte

d A

PD90

290.8

± 4.

031

7.3 ±

5.5

379.6

± 4.

048

7.4 ±

8.7

1010

080100

120

140

160

180

200

***

**

*

APD90 (% of baseline)

[E40

31] (

nM)

Veh

icle

(wat

er)

E40

31

1010

0020406080100

120

140

160

180

200

TRise (% of baseline)

[E40

31] (

nM)

Veh

icle

(wat

er)

E40

31

1010

080100

120

140

160

180

200

Cycle Length (% of baseline)

[E40

31] (

nM)

Veh

icle

(wat

er)

E40

31

0.1

60708090100

110

120

130

140

[Nife

dipi

ne] (

M)

Cycle Length (% of baseline)

Veh

icle

(wat

er)

E40

31

0.1

020406080100

120

140

160

180

200

[Nife

dipi

ne] (

M)

TRise (% of baseline)

Veh

icle

(wat

er)

E40

31

0.1

60708090100

110

120

130

140

***


[Nife

dipi

ne] (

M)

Veh

icle

(wat

er)

E40

31

0.1

60708090100

110

120

130

140

[Nife

dipi

ne] (

M)

***


Veh

icle

(wat

er)

E40

31

Bas

elin

e

200

ms

Dru

g

Bas

elin

e

200

ms

Dru

gB

asel

ine

200

ms

Dru

g

Bas

elin

e

200

ms

Dru

gB

asel

ine

200

ms

Dru

gB

asel

ine

200

ms

Dru

g

DM

SO (%

) 0

.003

0

.01

0

.03

Nif

.(M

) 0

.03

0

.1

0.3

N

if 0

.03

MN

if 0

.1M

Nif

0. 3

M

Cycl

e Le

ngth

, sec

Cont

rol

0.75 ±

0.08

0.73 ±

0.08

0.71 ±

0.08

Drug

0.62 ±

0.06

0.58 ±

0.07

0.87 ±

0.01

Dia

stol

ic p

erio

d,

sec

Cont

rol

0.46 ±

0.06

0.44 ±

0.04

0.43 ±

0.05

Drug

0.35 ±

0.04

0.3 ±

0.06

0.33 ±

0.06

APD

90, m

s

Cont

rol

287.8

± 26

.230

0.3 ±

39.3

284.5

±

31.0

Drug

288.4

± 57

.722

2.1 ±

20.2

218.1

±

17.9

Frid

eric

ia’s

Co

rrec

ted

APD

90

Cont

rol

317.2

± 20

.533

0.5 ±

32.1

317.4

±

24.4

Drug

335.5

± 55

.126

6.2 ±

15.1

262.5

±

19.1

Thi

s w

ork

was

sup

port

ed b

y th

e B

iote

chno

logy

and

Bio

logi

cal S

cien

ces

Res

earc

h C

ounc

il (U

K),

Cly

de B

iosc

ienc

es (

UK

) an

d A

xiog

enes

is.

M.P

. H-V

. is

reci

pien

t of

a p

ostd

octo

ral f

ello

wsh

ip f

rom

Fun

daci

on A

lfon

so M

arti

n E

scud

ero

(Spa

in).

Con

clu

sion

:Th

ese

resu

lts c

onfir

m t

he h

igh

sens

itivi

ty o

f op

tical

vol

tage

mea

sure

men

ts

appl

ied

to iP

SC-

CM

s, a

llow

ing

dete

ctio

n of

sm

all c

hang

es in

AP

char

acte

rist

ics

(<10

%)

and

supp

orts

the

use

of

this

tec

hniq

ue

during

dru

g sc

reen

ing.

PART 1. NHBE MODEL:

DEVELOPMENT:

CHARACTERISATION: Trans-Epithelial Electrical Resistance (TEER) and Junctions: Muco-Ciliary Epithelium:

APPLICATIONS:

NHBE model is fully-differentiated and stable between Days 24 – 33 in ALI culture

ZO-1 Occludin

Day

33

Day

1

TJ

D

AJ

600nm

C

B

PART 2. METABO-LUNG: INTRODUCING METABOLISM:

DEVELOPMENT:

Metabo-Lung allows in vitro metabolism of compounds “deposited” on the bronchial epithelium.

VALIDATION:

A. Graph displaying average TEER values of different NHBE donors over the culture period (Day 1 – 42). Donor A ( ); Donor B ( ); Donor C ( ). Error bars denote standard deviation; n = 3. The growth, plateau and demise periods have been identified for each key TEER period.

B. TEM image of NHBE culture denoting a Tight Junction (TJ), Adherens Junction (AJ) and a Desmosome (D).

-2000

0

2000

4000

6000

8000

10000

12000

14000

1 3 6 9 12 15 18 21 24 27 30 33 36 39 42

Day

TEER

(.c

m2 )

Growth Plateau

Demise

Isolation of

BE Cells

METHODS:

CONCLUSIONS

NHBE MODEL:

1. Morphologically the fully-differentiated NHBE model resembles the human in vivo bronchial epithelium

2. Developmental characterisation reveals that the NHBE model is stable and fully-differentiated between Days 24 – 33 (Exposure Window)

3. The NHBE model contains: basal, intermediate, serous, Clara, goblet and ciliated cell types

4. Highly cost effective; 1 cryovial (~0.5million cells) yielding ~350 24-well size cell culture inserts at a cost of ~£2,500

METABO-LUNG MODEL:

1. Primary human hepatocytes enables in vivo-likemetabolism of compounds resulting in multi-organ relatedtoxicity data for ‘inhalable’ compounds

2. Biotransformation data is a valuable tool for the development of safer medicines

3. Innovative opportunity to Replace, Refine and Reducethe use of animals in research and safety testing

ABSTRACT: The respiratory tract is the primary site of exposure to airborne substances and the bronchial epithelium is one of the first lines of defence. Airway diseases such as, asthma and chronic obstructive pulmonary disorder (COPD) cause disruption and/or re-modelling of the bronchial epithelium. Lung diseases and their burden are on the increase and therefore the principle focus of respiratory medicine is to gain better understanding of lung injury/repair mechanisms and the development of more effective therapeutics. Traditionally, animal models have been the primary model in respiratory research; however, they are deficient in many key areas in their ability to mimic the human response to inhaled compounds. A growing need therefore exists for an accurate, in vitro model of the bronchial epithelium which accurately reflects the human in vivo situation. We have developed a normal human bronchial epithelial (NHBE) model of the bronchial epithelium cultured at an air-liquid interface (ALI). This NHBE model is a fully-differentiated, pseudo-stratified, muco-ciliary epithelium containing basal, serous, Clara, goblet and ciliated cells. The NHBE model is stable and fully developed form Days 24 – 33 in ALI culture. A further development was to co-culturing the NHBE model with primary human hepatocytes, creating the metabolising bronchial model; the Metabo-Lung. The co-culture of bronchial and liver cells provides us with a multi-organ response to any inhaled compounds tested. The addition of metabolising capabilities to the NHBE model will result in more accurate inhalation toxicology outcomes for testing: 1) ambient air particles (e.g. air pollution); 2) candidate respiratory drugs (e.g. for COPD); 3) candidate drugs with the lung as a delivery method; 4) toxicity testing of respirable compounds.

3RS BENEFITS:

Basal Cells (Progenitors) Proliferation

(Progenitors)

NHBE MODEL

Differentiation

ALI Culture

BC

CCGC

ICClC

10μm

Basal Cell Goblet Cell(Releasing Mucus)

IntermediateCell

Ciliate Cell Serous Cell Clara Cell

Cells of the NHBE Model:

A A

1μm

Cilia

BasalBody

5μm

Mu&

Gx

600nm

C

B

D

Cell Culture Insert

MicroporousMembrane

24-Well Plate

Media

Patent:WO2012/123712

Toxicological Analysis

Morphological Analysis

Genomic Analysis

Compound/Bacteria

Incubate

x hours

PBS Wash

Prytherch et al (2011) Macromol. Biosci. 11; 1467

LiverLung METABO-LUNG™

Testing CYP1A2 (Phenacetin) and CYP3A4 (Midazolam) Activity in Hepatocyte, NHBE and Metabo-Lung Models

A panel of compounds which are metabolised by 8 different CYPs, as well as a compound which undergoes Phase I and Phase II metabolism are to be screened in all three models

Post-incubation with each primary compound the following analysis will be undertaken:

Culture viability HPLC detection of metabolite/sMorphology

1. NHBE MODEL – Undifferentiated NHBE cells (0.5 million cells;

Lonza, Switzerland) were subcultured in flasks– Subcultured NHBE cells were seeded into

6.5mm diameter Millipore® 24-well cell culture inserts (Day 0)

– Cultured at an air-liquid interface (from Day 1) – Extensive characterisation (TEER, LM, TEM,

SEM, IHC) undertaken every three days – NHBE exposure window between Days 24 - 33

2. METABO-LUNG MODEL – NHBE model cultured as above – Hepatocytes cultured in 24-well collagen

coated plates – Fully-differentiated NHBE inserts transferred to

form Metabo-Lung co-culture

Both the NHBE and Metabo-Lung Models arederived from primary human cells and therefore can be used as absolute REPLACEMENTS for animal models

Integration of the NHBE and Metabo-Lung Models into current toxicological and drug development protocols could help REDUCE andREFINE the use of animal models

C. Tight junction specific proteins ZO-1 and Occludin; sparse cytoplasmic presence on Day 1, followed by migration and accumulation at cell-cell borders from Day 15 (Day 33 shown).

C. TEM image of the apical region of a ciliated cell; presence of cilia and anchoring basal bodies. Inset; cross-section of cilia displaying 9+2 axoneme formation.

D. TEM image of the apical region of a ciliated cell displaying mucin and glycocalyx between the cilia.

A. Toluidine blue stained LM section of the NHBE model. The epithelium is pseudo-stratified and contains, basal (BC), intermediate (IC), goblet (GC), ciliated (CC) and Clara (ClC) cells.

B. SEM image; surface topography of the NHBE culture.

IN VITRO NHBE MODELS OF THE HUMANBRONCHIAL EPITHELIUM: THE FUTUREOF INHALATION SAFETY ASSESSMENT

ZOË PRYTHERCH1& KELLY BÉRUBÉ1

1 Cardiff School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3A

• Primary compound is metabolised in Metabo-Lung model – creating an in vitro metabolising bronchial model

-5.0

0.0

5.0

10.0

15.0

20.0

25.0

30.0

0 100

Ace

tam

inop

hen

(M

)

Phenacetin ( M)

Hep Alone NHBE Alone Co-Culture

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 100

1-H

ydro

xym

idaz

olam

(M

)

Midazolam ( M)

Hep Alone NHBE Alone Co-Culture

6. Blood brain barrier 4. Myelination

3. Targeted treatments

2. Aligned tissue models

( III-tubulin MBP Hoechst)

1. Reactive gliosis

5. 3D Cellular interfaces

150 m

Tenofovir abolishes Na+-dependent phosphate uptake in human proximal tubule cell monolayers. Sarah Billington*, Git Chung, Colin Brown

Institute for Cell and Molecular Biosciences, Newcastle University Email: [email protected]

Introduction

Methods

Discussion

Understanding the impact of drug molecules upon kidney function has been restricted by the lack of suitable in-vitro models of human kidney. To address this, we have developed highly predictive in-vitro human primary proximal tubule cell (PTCs) models to investigate the interactions of drug molecules with proximal tubule cells. Here we demonstrate the power of human PTCs by identifying the mechanism by which tenofovir disrupts renal phosphate handling. Tenofovir has been associated with hyperphosphaturia, hypophosphatemia and the onset of osteomalacia in around 30% of patients. These adverse effects have been attributed to the renal toxicity of tenofovir and decline in proximal tubule function.

Kidney decapsulated, cortex dissected and finely chopped.

2 hour collagenase digest in isolation medium at 37 C.

The heterogeneous cell population was passed through 40uM sieve, then

separated by density centrifugation.

The tubular cell layer was extracted and cultured on Transwell inserts as shown

in Figure 2.

Figure 1: PTC isolation

Figure 2: PTCs cultured on Transwell insert for uptake studies

Results

Characterisation of the sodium dependent phosphate transporter within human primary tubular cells. To confirm the presence of the sodium dependent phosphate transporter (NaPi-2a) within human PTCs, monolayers were incubated with 100 M [32P]-phosphate in the presence or absence of sodium (137 mM or 0 mM, respectively) over a range of incubation times. Linear regression analysis of the data shows that the reabsorption of [32P]-phosphate by human PTC monolayers was abolished in the absence of sodium.

Retention of [32P]-phosphate within human primary tubular cells. To determine the pharmacokinetics of [32P]-phosphate intracellular retention, monolayers were incubated with various concentrations of [32P]-phosphate for 5 minutes. Non-linear regression analysis of the data gave an apparent Vmax of 458.10 ± 8.26 pmol of [32P]-phosphate / min / cm2 and a Kd value of 64.50 ± 4.13 M [32P]-phosphate.

Results

Initial experiments demonstrated the expression of the renal sodium-dependent phosphate transport (NaPi-2a) on the apical membrane of human PTC monolayers. This transporter is the key transporter mediating the reabsorption of phosphate from the filtrate into the blood. The uptake of [32P]-phosphate across the apical membrane of human PTCs was both Na+-dependent and saturable with an apparent Kd of 64.50 ± 4.13

M. Importantly, the uptake of [32P]-phosphate (100 M) was completely abolished by the addition of tenofovir at the apical membrane. Tenofovir inhibited phosphate uptake with an apparent Ki value of 66.3 ± 0.9 M.

Inhibition of [32P]-phosphate reabsorption across the apical membrane by tenofovir. To determine the pharmacokinetics for the inhibition of phosphate reabsorption by tenofovir, monolayers were incubated with 100 M [32P]-phosphate in the presence of various concentrations of tenofovir for 5 minutes. The mean Kd value was 66.63 ± 0.91 M tenofovir. Using the Cheng – Prusoff equation the estimated IC50 value would be 166.58 M.

The impact of tenofovir on the intracellular reabsorption of [32P]-phosphate across the apical membrane. To identify if tenofovir had an affect upon tubular reabsorption of phosphate, monolayers were incubated with 100 M [32P]-phosphate in the presence and absence of 10 M Tenofovir. Tenofovir significantly reduced tubular reabsorption of [32P]-phosphate at the apical membrane of human PTC monolayers (P < 0.0002). Significance was assigned using Students t-test.

PTCs were isolated from human kidney as described in Figure 1 less than 18 hours ex vivo. Donors are males and females between 0 and 76 years old.

To measure monolayer formation and tight junction integritytrans-epithelial electrical resistance (TEER) was measuredthroughout incubation at regular time intervals using anelectric voltohmeter. Experiments were performed whenhuman PTC monolayers had a TEER of 80 .cm2 or greater.

Figure 3: Tight junction integrity of human PTC monolayer

Conclusions

Our data suggest that the impact of tenofovir upon renal phosphate handling does not arise from tenofovir-induced proximal tubule damage but rather from an inhibition of NaPi-2a mediated phosphate uptake. The abolition of phosphate uptake by tenofovir results in hyperphosphaturia and subsequently impacts plasma and bone phosphate levels. The data highlight the importance of developing holistic cell based models of the human proximal tubule. The novel mechanistic understanding of tenofovir/phosphate interactions has been achieved in vitro without resorting to the use of animals.

Practical considerations of using human gastrointestinal tissue for physiological experiments

Introduction

Obtaining human tissue from surgical resections

>70 items of patient history collected

Several hospitals are used for tissue supply

Tissue obtained from throughout GI tract

Contracti

on Durin

g EFS

Rlaxati

on Durin

g EFS

Contracti

on Afte

r EFS

0

20

40

60

80

100

% o

f str

ips

No change in the response phenotype to EFS after overnight storage in colon

0

100

200

300

400

time

(min

utes

)

Tissues take longer to achieve stable EFS-evoked responses after overnight storage

Conclusion

Tissue suspended in tissue baths for electrical field stimulation (EFS) and isometric recording of neuromuscular responses

EFS evokes different phenotypes of response in human colon muscle strips

Functional Studies with Human Gastrointestinal Tissues

WHY IS THIS SO IMPORTANT? USING HUMAN GI TISSUES TO STUDY ADVERSE EVENTS

USING HUMAN GI TISSUES TO DEVELOP NEW DRUGS

CONCLUSIONS

Nausea

Diarrhoea

-8 -6 -4

-50

0

50

100

150

log10[agonist]

% increase frombaseline EFS

-8 -6 -40

500

1000

1500

log10[agonist]

% increase frombaseline EFS

Stimulants of gastric emptying with different efficacies in human gastric

antrum6

Kappa opioid receptor agonists with similar predicted efficacies but different efficacies

in human colon: Biased agonism?5

-10 -9 -8 -7 -6 -5-50

0

50

100

150

log10[agonist]

% E

FS

mag

nit

ud

e

-10 -8 -6 -4

-50

0

50

100

150

log10[agonist]

% E

FS

mag

nit

ud

e

asimadoline

ICI204448

20 40 60 80 1000

50

100

Patient Age (years)

% of stripsthat relax

during EFS

EFS10 s

EFS10 s

EFS10 s

EFS10 s

Predicting regional differences in how different motility stimulants are effective7

Long term 3D human liver microtissue co cultures:characterization and implication for drug induced hepatotoxicity studies

Day 7 Day 14 Day 21 Day 28

H&E

CD68

100 m

LPS

+ LPS

Native human liver 3D Human liver microtissues

BSEP

Morphology of 3D human liver microtissue co cultures with Kupffercells

Kupffer cell functionality of human liver microtissues

Viability and hepatocyte functionality of human livermicrotissues

R&D - SEACSafety & Environmental Assurance Centre

Use of human ex vivo skin in safety riskassessment of cosmetic ingredients.Ruth Pendlington, Safety & Environmental Assurance Centre, Unilever,

Colworth Science Park, Sharnbrook, BEDFORD, MK 44 1LQ, UK

Introduction

Date: 30/07/2014

Assessment of exposure

Challenges

• Ability to routinely source ex vivo human skin; reproducibility in these experiments can be an issue due to donor variability, such that typically n = 12+ cells arerequired for data generation for safety risk assessment.

• Ensuring that this work complies with all appropriate safety, ethical, legal, and Corporate requirements.• Working with our Local Research Ethics Committee to ensure that commercial suppliers:

•adhere to the requirements of the Human Tissue Act (if tissue is sourced in the UK) or with other national regulations if sourced elsewhere•provide documentation that demonstrates informed consent for use in non-medical research has been obtained.

References[1] OECD Guideline for Testing of Chemicals, Guideline 428: Skin Absorption: In Vitro Method (2004).[2] SCCS/1358/10 Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients (2010) http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_s_002.pdf

0

5

10

25

30

35

40

45

50

Stratum corneum Epidermis Dermis Receptor

Rec

over

y (%

)

Distribution of ingredient X in dermatomed human skin after application for 24hr in a skin lotion

Formulation 1 Formulation 2

0.00

0.05

0.10

0.15

0.30

0.35

0 4 8 12 16 20 24

Flux

(g/

cm2/

hr)

Time (hr)

Flux of ingredient X through dermatomed human skin


Figure 2: Example data

Symbol Term UnitsSEDd Systemic Exposure Dose for dermal route g/kg

C Percentage of Ingredient in Product %A Amount of Face Cream Used per Day g/dayRF Retention Factor 1 (leave on product)DA Skin Penetration %BW Body Weight kg

Consumer safety risk assessments for cosmetic ingredients are always exposure-driven. For dermally applied ingredients, risk assessments require anunderstanding of the kinetics of ingredients following consumer exposure via the skin. Since the mid-1990s, we have been using a model of in vitro (ex vivo) skinabsorption to assess the potential for an ingredient to be absorbed through the skin, in place of in vivo toxicokinetic studies in rats. This method, althoughpreceding the OECD guidelines 428 [1] which were issued in 2004, conforms to these guidelines. The Scientific Committee for Consumer Safety (SCCS) (theScientific Committee that provides the European Commission with opinions on health and safety risks of non-food consumer products) regard ex vivo human skinas the gold standard for skin absorption studies and have their own set of guidelines for the cosmetic sector [2]. The methods used and challenges to thisapproach are described below

SEDd = ((C/100) x A x RF x (DA/100) x 1000000)BW

The formula used to calculate the systemic exposure to ingredients present in face creamvia the dermal route is as follows:

Regarded as not absorbedTest item:• Rinsed from the skin surface• Swabbed from the skin surface• Associated with the outer clampedskin

• Associated with the cell wash

Regarded as absorbedTest item:• Penetrating into the receptor solution• Remaining within the skin at the

terminal time point

DebatableTest item:• Associated with the tape strips,

determined by interpretation of thetape strip profile

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Rec

over

y (n

g eq

uiv

/cm

2)

Tape strip

Distribution of ingredient X through stratum corneum of dermatomed human skin


(c) Schematic of standard method

Tape strip inner skinsurface

Digest epidermis,dermis & outer skinsamples

Excise inner dosed area of skinfrom outer clamped area

Disc of human split thicknessskin cut out and mounted in cell

Dose applied Collect receptorsolution

Wash skin surface of n = 4 cells at0.5, 1, 2, 4, 8 & 24hr

then remove skin from cell

(d) Schematic of time course method

Disc of human split thicknessskin cut out and mounted in cell

Dose applied Collect receptorsolution

Wash skin surface at 24hrthen remove skin from cell

Wash skin surface@ intermediatetime point (optional)

Continue to collect receptorsolution in hourly fractions

Excise inner dosed area of skinfrom outer clamped area

Tape strip inner skinsurface, heat separateepidermis from dermis

Digest epidermis,dermis & outer skinsamples

Figure 1: In vitro skin absorption methods and equipment

Receptor solutionreservoir

Peristaltic pump

Fraction collector

Heated cell holderDiffusion cells

Sample collectionvials

(a) Flow-through diffusion cell

(b) Equipment set up

PositionPositionof skinof skin

Date: 15/07/14

Click t

o buy NOW!

PDF-XChange

www.docu-track.com Clic

k to buy N

OW!PDF-XChange

www.docu-track.com

Background:

Genetic damage can lead to the development of cancers. Agents that damage DNA are called genotoxins.

mutagens: alter the DNA sequenceclastogens: break chromosomesaneugens: cause chromosome mis-segregation pro-mutagens: produce genotoxic metabolites

There are long-established genotoxicity tests, and innovation is rare in the regulatory arena. As problems have been identified, protocols have evolved,

Ames reverse mutation test. Uses bacterial mutantsdependent on amino acid supplements. Reverse mutations allow colony formation without supplements.

- +Comet assay. Clastogens break chromosomes. The fragments move more quickly through gels, producing characteristic ‘comets’.

Micronucleus test (MNT). Chromosome that mis-segregate properly, or chromosome fragments can be packaged into “micronuclei”.

Sensitivity% Specificity% Concordance%AssayAmes 60 88 74MNT 90 66 72MLA* 87 80 84GADD45a 88 96 92 (Data from 154 compounds)

*in vitro Mouse Lymphoma Assay (MLA). This is the least accurate of the in vitro,assays. A recent data review by its most staunch defender (FDA’s Martha Moore) foundwide protocol variations, and deemed the majority of published data unreliable. Thereis a clear need for better more reliable assays.

Human cell lines in genotoxicity assessmentRichard M WALMSLEY

Gentronix Ltd & University of Manchester Faculty of Life Sciences. UK

Table 1:Prediction of carcinogenicity by in vitro genotoxicity assays

A positive result

GFP

RSG

A negative result

HumanTK6 cells containing the reporter are exposed to test compounds for 48h (3hplus 45h recovery in S9 protocol). Data collected by spectrophotometry or flowcytometry. Reporter output is normalised to cell numbers to produce a brightnessvalue: increase of 1.5 fold over control defines positive result. Data are rejectedbelow 30% RSG.

Summary.

1. Human derived TK6 cells are the basis for a validated and accurate, pan-genotoxin hazard assessment assay.

2. Human derived MCL-5 cells may be the basis for a new, and better gene mutation assay*.

1. Human cell “GADD45a” assay: identifies mutagens, aneugens

and clastogens

GADD45a has a key role in the response to genome damage. Published studies demonstrate high sensitivity and specificity. Its low compound requirement has led to adoption in genotoxicity hazard/safety screening in diverse sectors. Higher accuracy regulatory in vitro tests also contribute to weight of evidence arguments, where other in vitro test results are conflicting.

Abstract:

Detection of putative genotoxic carcinogens is an important aspect of chemical safety assessment.

Many of the rodent cell lines used in genotoxicity assessment have mutations in the p53 gene that affect DNA repair, and produce too many positive results for non-carcinogens. As a result, many animals (mouse/rat) are often required to resolve data conflicts between in vitro test results.

This poster describes the use of two human lymphoblastoidcell lines with functional p53 genes, in two different assays:

1. The extensively validated and widely used GADD45a-GFP “GreenScreen HC genotoxicity screening assay, which accurately identifies all classes of genotoxins.

2. The developing “Pig-A” assay that specifically identifies eukaryote mutagens.

2. Human cell “Pig-A” assay: only identifies mutagens,

but no pigs required!

The Pig-A gene is needed to make “GPI anchors”, which anchor proteins to the surface. Fluorescently tagged antibodies distinguish normal cells from Pig-A mutants (which have no anchors), so anchor mutants can live and be counted.

There is now an animal Pig-A assay. A human cell Pig-A assay could predict & reduce animal testing.

Anchored protein

Cell membrane

Tagged antibody

GPI anchor

Tk6Parent Pig-A wild type

CD59 +vePig-A mutant

CD59 -ve

Pig-A sample data

b. We ‘sorted’ them into distinct collections ofwild type and mutant cells.

However, we also found that our favourite human TK6 cell data were notvery reproducible. We are now progressing with a new favourite human cell line, MCL-5

GADD45a regulatory elementsdrive GFP/luciferase synthesis.

p53 element

GADD45a reporter sample data

c. Wild type cells showed EMS-induced increase in Pig-A mutants.

a. Our favourite human TK6 cell culture was a mixture of wild type and mutants.

Ex vivo in vivo

in vivo

J Clin Patholet al.

RegulatoryToxicology and Pharmacology

Impact of iPS Cell Technology on Animal Reduction for Drug Discovery and Disease Research

Giorgia Salvagiotto, Dominic Hussey, Arne Thompson, Susan DeLaura, Blake Anson, David Mann, and Vanessa Ott

www.cellulardynamics.com Madison, WI USA +1 (608) 310-5100

Abstract

Predictive Human System for Cardiac Arrhythmias Problem:

iCell-based solution:

Summary

sensitivity predictivity biological relevance

Target Identification

TargetValidation

CompoundScreening

LeadOptimization

PreclinicalTrials

ClinicalTrials

Relevant Human System for Infectious Disease Modeling

Problem:


SR-BICD81

HCV Attachment

A B Claudin 1 Occludin

HCV Entry

C D

Adult Primary Hepatocytes iCell Hepatocytes

SYMBOL Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3OCLNCLDN1CD81SR-B1

Problem:


Sensitive Cell-based Alternative to Mouse Bioassay

iCell Neurons RSC cells

0 0

Uncleaved

Cleaved

Toxin Detection Toxin Neutralization

iCell Hepatocytes are susceptible to HCV infectivity

Qualitative Responses Investigative Toxicology Predictive Screening

xCELLigence and MEA reading of RO5657-treated iCell Cardiomyocytes

Comparable Performance with Accepted Assays (i.e., Rabbit Wedge)

iCell Neurons responses to BoNT are highly sensitive

Human tissue cover 2014 Tissu… · CV T CV L Electrophysiology: Conduction Velocity Pharmacology: E-4031, Chromanol 293B and 4-AP A C E-4031 Figure 6 (IIHFW RI WKH ,.U LQKLELWRU(

Documents