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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
***
APD75 (% of baseline)
[Nife
dipi
ne] (
M)
Veh
icle
(wat
er)
E40
31
0.1
60708090100
110
120
130
140
[Nife
dipi
ne] (
M)
***
APD50 (% of baseline)
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
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Thi
s w
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e B
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and
Bio
logi
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Res
earc
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Cly
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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
Formulation 1 Formulation 2
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
Formulation 1 Formulation 2
(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
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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:
iCell-based solution:
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:
iCell-based solution:
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