« IN VITRO MODELS OF THE BLOOD-BRAIN
BARRIER AND THEIR USE IN DRUG
DEVELOPMENT »
Alicante
eSI Meeting, Sept. 29-30
Pr Roméo Cecchelli
The ageing of the population gives to
neurodegenrative diseases a
frightening future because they relate
to a population more and more
important
But
95 % of new synthetised molecules do
not reach the brain….
WHY ?
Ehrlich 1885
Goldmann 1913
The BBB was located in brain capillaries
The lenght in Human is around 650 km
Its surface area is around 12 m2
The mean distance between 2 capillaries is around 40 microns
• Brain capillaries are a complex
structure
Brain capillaries
Endothelial cells
Astrocytes
Astrocytes
Basal lamina
Pericyte
Endothelial cell
The Blood-Brain Barrier Cellular and Molecular Biology
William M. Pardridge 1993
The Blood-Brain Barrier Cellular and Molecular Biology
William M. Pardridge 1993
BBB : a physical barrier
Blood
Apical plasma membrane
Tight
junction
claudins
ZO-1
ZO-1
ZO-1
ZO-2
ZO-1
AF6
ZO-1
ZO-1
JAMs
ZO-3
occludin
ZO-3
/
Cadherins
Cadherins
Adherens
junction
Brain
cingulin
ZO-2
/
7H6
Actin
Peripheral capillary
Endothelial Cell Biology
N. Simionescu and M. Simionescu 1988
Peripheral capillary
Endothelial Cell Biology
N. Simionescu and M. Simionescu 1988
BBB: PHYSICAL BARRIER
blood
tight
junction
endothelial cell
brain
Low transcellular
transport
No paracellula
passage
BBB : a metabolic barrier
Blood
L-DOPA
P-gp
MAO
Drug-metabolizing
enzymes
L-DOPA
Dopamine
Degradation
Brain
BBB : A METABOLIC BARRIER
UGT-1A6
GST
Pericyte
MAO-B
Glutamyl
aminopeptidase
CYP 450
CYP 1A1 (rat)
CYP 1B1 (human)
CYP 2B1 (rat)
CYP 2B6 (human)
Endothelial cells
Transport processes through a cerebral
endothelium
Blood
Specific transport
(receptor or transporter)
«fluid-phase»
transcytosis
Paracellula
pathway
ZO
ZA
Brain
Different techniques used to study the BBB
3H/14C-labelled
drug
(to measure BBB
permeability)
14C/3H
–sucrose/inulin
(to measure cerebrovascular volume
RESEARCH
1 to 2 years
1 to 2 years
SEARCH
OPTIMISATION
FOR
CHEMICAL
STARTING
POINTS
DEVELOPMENT
1 to 2 years
IN VITRO &
ANIMAL
PRECLINICAL
EVALUATION
• Potency
•Conception
• Selectivity
•Synthesis
• Oral bioavailability
• Duration
of action
SCREENING
• Activity
• Characterisation
• Stability
• Safety
• Efficacy in
whole
organism
6 to 8 years
CLINICAL HUMAN EVALUATION
PHASE
I
PHASE
BIRTH
LIFE
1 to 2 years
FILE
SUBMISSION/
REGISTRATION
Tolerance
II
MARKETING
&
APPROVAL
pharmacoBiological
PHASE
kinetics
Activity
III
&
research
Confirmation
of a
of safety
thera&
peutic
therapeutic
effect
effect
PHASE
IV
In vivo techniques
These techniques can not be used in screening to evaluate
the brain penetration of a molecule
In vitro methods
Cell lines
• All immortalized brain capillary endothelial cell
lines (RBE4, MBEC line, TR-BBB ) do not
expressed must of the tight junction proteins
and are very leaky.
• They cannot be used to study transcellular
transport.
I just want to focus my presentation on primary
and long term culture of brain capillary
endothelial cells.
Classical Method
Gray matter
Enzymatic digestion
+
Centrifugation
Microvessel endothelial cells and pericytes
Capillaries
Arterioles
Venules
Pericytes
Primary culture of microvessel endothelial cells
Gray matter
Mechanical homogenization
+
Filtration
Isolated microvessels
Arterioles
Capillaries
Capillaries
Veinules
Extracellular Matrix
Pure capillary endothelial cells
What about the Blood Brain Barrier
marker ?
Freeze fracture examination of endothelial cells
Transendothelial electrical resistance = 400 Ohms.cm2
Histochemichal detection of g-GT in brain capillaries
Units / (mg prot.min)
prot.min)
Gamma-GT expression
40
0
100
100
+
+
x
x
100
100
100
100
100
100
+
+
+
+
+
+
x
x
x
x
x
x
300
F VIII
% positive cells
Tight
junctions
MAO
ACE
200
100
gamma-GT
0
Isolated P1
capillaries
P
2
P3
P4
BBCE
P
5
P6
P
7
Conclusion
Consequently, endothelial cells in culture
alone can not be considered as a relevant
blood-brain barrier model
The BBB localisation
Astrocytes
Endothelial cell
The in vitro BBB model
Blood
Endothelial cells
Brain
Glial cells
Glial cell repartition
Astrocytes
Oligodendrocytes
Microglia
What about the g–GT activity ?
Histochemichal detection of g-GT in brain
capillaries endothelial cells
Statistical study of 2D-PAGE area from BCECs
Co-culture
Solo-culture
Important variation : overexpressed in co-culture
No statistical significant variation
Tight Junctions
Blood
Apical plasma membrane
Tight
junction
claudins
ZO-2
ZO-1
cingulin
ZO-2
ZO-1
ZO-1 ZO-3
ZO-1
occludin
ZO-3
AF6
ZO-1
ZO-1
JAMs
/
Cadherins
Cadherins
Adherens
junction
/
7H6
Actin
The transendothelial electrical resistance raises
from 400 Ohms.cm2 in soloculture
to 800 Ohms.cm2 in coculture
k Da
205
140
BB
co CE
cu
ltu in
re
BB
CE
cu in
ltu so
re lo
I
ca sola
pi
lla ted
rie
s
P-gp detection by Western Blot
analysis
Analysis of OCTN2 and P-gp immunofluorescent staining
in apical and basolateral membrane of endothelial cells
Fluorescence intensity
OCTN2
P-gp
nuclei
30
20
10
0
0
5
10
15
Slides (1 = 0.16μm)
24μm
Apical membrane
Basolateral membrane
Inhibition study (Uptake)
500
%/control
400
VINCRISTINE
300
200
100
0
Control
S9788 (1M)
Conclusion
In coculture with astrocytes, brain capillary
endothelial cells present most of the
characteristics that are known to have
important functions in vivo
How can we use this model to predict the
drug brain penetration ?
General working process
Refreshing glial cells medium (twice a week)
Preparation of glial cells culture medium
Refreshing BCEC medium
(every 2 days)
Defrosting glial cells vial
Preparation of BC EC culture medium
60mm dishes coating
Defrosting BC EC vial
Filters coating
Trypsinisation
S eeding
Transport experiment
Analytics
Raw data
processing
3 weeks
Glial Cells
12 days
BCEC
Co-culture
Experiments
BBB Permeability studies
T + 15 min
T + 15 min
Dosage
1000
SUCROSE
900
Pe = 0,24 x 10
800
-3
cm/min
clearance in L
700
PSf = 14,38 L/min
600
R
500
2
= 0,99
400
300
PSt = 0,95 L/min
200
R
100
2
= 0,99
0
0
10
20
30
40
time in min
50
60
70
1200
CAFFEIN
clearance in l
1000
Pe = 59,60 x 10
-3
cm/min
800
PSf = 16,16 L/min
600
2
R = 0,99
PSt = 15,18 L/min
400
R
2
= 0,99
200
0
0
10
20
30
40
Time in min
50
60
70
Correlation between permeability values obtained in vivo with the in vivo
techniques (Brain perfusion and Oldendorf) and in vitro with the BBB
model
Oldendorf’s Technique
Cerebral Perfusion
9
-1
Nicotine
-2
Propranolol
7
Phenytoin
6
Caffeine
5
Dopamine
4Acetylsalicilic
3
Sucrose
Acid
Hydrocortisone
Urea
2
-10
R = 0.93
-9
-8
-7
-6
In Pe x MW (cm/s)
In vi tro BBB Model
In Pe x MW (mg/mi n)
In vi vo
In BUI x MW
In vi vo
8
Diazepam
Antipyrin
-3
Hydrocortisone
Manitol
-8
-9
-10
-11
Urea
Sucrose
-9
-5
R = 0.98
Propranolol
Phenytoin
-4
-5
-6
-7
Caffeine
-8
-7
In Pe x MW (cm/s)
In vi tro BBB Model
-6
-5
In Silico / In vitro correlation
Correlation between BBB cell culture
permeability and in silico prediction
R2 = 0.52 => R =
0.72
Q2 = 0.50
Compounds measured
by Stefan Lundquist
and Mila Renftel
Research DMPK,
Södertälje. In silico
model developed by
Markus Haeberlein,
Chemistry Dept,
Södertälje
bbp03M21
Reproducibility
- 6 last months
- 3 different clones (B1, PK, Lau1) between P4 et P7
- 4 different technicians
Pe (sucrose) = 0,28 ± 0,12 (n=81)
Astrazeneca (Sweden)
Pe (sucrose) = 0,32± 0,18 (n=52)
Ranking
Example of product
Pe (x 10-3 cm/min)
Caffeine
59.90
Nicotine
58.76
Diazepam
19.86
Antipyrine
18.74
DiPhenylHydantoine
7.37
Urea
2.22
Morphine
1.90
Verapamil
1.74
Warfarin
1.72
L Dopa
1.29
Vinblastine
1.19
Alanine
1.07
Leucine
0.91
Lactic acid
0.63
Sucrose
0.58
Glycerol
0.44
Cyclosporin
0.42
AZT
0.39
Cimetidine
0.26
Digoxin
0.21
Vincristine
0.15
Inulin
0.04
Brain penetration
VERY GOOD
30.10-3 cm/min
GOOD
2.10-3 cm/min
LOW
1.10-3 cm/min
VERY LOW
This in vitro model constitutes a
RELEVANT model to the BBB
RESEARCH
1 to 2 years
1 to 2 years
SEARCH
FOR
CHEMICAL
STARTING
POINTS
OPTIMISATION
DEVELOPMENT
1 to 2 years
IN VITRO &
ANIMAL
PRECLINICAL
EVALUATION
• Potency
•Conception
• Selectivity
•Synthesis
• Oral bioavailability
• Duration
of action
SCREENING
• Activity
• Characterisation
• Stability
• Safety
• Efficacy in
whole
organism
BIRTH
6 to 8 years
1 to 2 years
CLINICAL HUMAN EVALUATION
PHASE
I
PHASE
Tolerance
II
&
pharmacoBiological
kinetics
Activity
&
research
of a
therapeutic
effect
LIFE
FILE
SUBMISSION/
REGISTRATION
MARKETING
APPROVAL
PHASE
III
Confirmation
of safety
&
therapeutic
effect
PHASE
IV
General working process
Refreshing glial cells medium (twice a week)
Preparation of glial cells culture medium
Refreshing BCEC medium
(every 2 days)
Defrosting glial cells vial
Preparation of BC EC culture medium
60mm dishes coating
Defrosting BC EC vial
Filters coating
Trypsinisation
S eeding
Transport experiment
Analytics
Raw data
processing
3 weeks
Glial Cells
12 days
BCEC
Co-culture
Experiments
General working process
4D@Screen Pack
1 day : C ELLIAL BBB-inducing medium
Preparation of BC EC culture medium
60mm dishes coating
Defrosting BC EC vial
Filters coating
Trypsinisation
Seeding
Transport experiment
Analytics
Raw data
processing
4 days
Preparation of
in vitro BBB model
Experiments
General working process
Refreshing glial cells medium (twice a week)
Refreshing BCEC medium
(ev ery 2 day s)
Preparation of glial cells culture medium
Defrosting glial cells v ial
Preparation of BCEC culture medium
60mm dishes coating
Defrosting BCEC v ial
Filters coating
Try psinisation
Seeding
CT Bovial@Screen Pack
12 day s
3 weeks
Glial Cells
Transport experiment
Analy tics
Raw data
processing
BCEC
Co-culture
Experiments
1 day : CELLIAL BBB-inducing mediu
4D@Screen Pack
Transport experiment
Analy tics
Raw data
processing
4 day s
Preparation of
in v itro BBB model
Experiments
Culture Time
CT Bovial@Screen Pack
/ 4D@Screen Pack
3 weeks (glial cell culture)
Co-culture
+ 7 days (confluence)+ 5 days : BBB ready
60mm-culture dish
4 day-culture
4 days : BBB ready
BBB characteristics
4D@Screen Pack
Cell monolayer morphology:
Vimentin
Bar = 25 μm
Actin
BBB characteristics
4D@Screen Pack
Tight
junctions:
Bar = 25 μm
Occludin
ZO-1
Claudin-1
Claudin-5
Control of BBB integrity
Sucrose permeability
4D@Screen Pack
Integrity threshold
12 dayCo-culture
4 dayculture
P-glycoprotein
4D@Screen Pack
Presence of the protein
(Western Blot)
MW
(kDa)
220
94
66
Functionality of P-gp
(inhibition assay)
Transporters (mRNA)
RT-PCR from co-cultures, capillaries and 4 day-endothelial cells
coc
C
4 day-e
coc
C
4 day-e
P-gp
MRP1
MRP4
MRP5
MRP6
- actin
Seeding
CT Bovial@Screen Pack
/ 4D@Screen Pack
90 filters
12 day-system
6 well-filters
1 vial of
endothelial cells
3 x 60mm-culture dishes
226 filters
4 day-system
24 well-filters
Correlation
CT Bovial@Screen Pack
/ 4D@Screen Pack
Correlation between in vitro BBB (24w-4d permeability)
and in vivo BU (brain perfusion)
Ln[PexVMW] (ml/g/min) BU
10
All parameters were normalized for molecular weight
9
8
7
6
5
4
2
R = 0.8538
3
2
1
0
-4
-5
-6
-7
-8
Ln[PexVMW] (cm/s) in-v itro BBB
-9
-10
-11
Correlation
CT Bovial@Screen Pack
/ 4D@Screen Pack
Correlation between in vivo BU (brain perfusion)
and in vitro MDCK (Papp)
Ln[PexVMW] (ml/g/min) BU
All parameters were normalized for molecular weight
10
2
R = 0.3956
9
8
7
6
5
4
3
2
1
0
-6.00
-7.00
-8.00
-9.00
-10.00
-11.00
-12.00
-13.00
-14.00
Ln[PexVMW] (cm/sec) MDCK
-15.00
How to regulate, dose, improve the
cerebral transport ?
By testing hits or leads on the in vitro model to have an experimental
result of reference for permeability
By testing optimized compounds ( affinity , selectivity ) on the in vitro
model to check the increase in permeability and select the best
canditates
By testing selected compounds on in vivo model to check the in
vivo cerebral penetration
The in vitro model is an complementary tool
which provide data to help for decisions
It allows mechanistic studies on a cellular and a molecular
level (Pgp- Specific transporters, pathological conditions
i.e. stroke, inflammation)
Use of the transcytotic pathway
Blood
Low density lipoproteines (LDL)
ZO
ZA
Brain
Conclusion
ZO
Lamp 1
Légèrement acide
ZA
Nanoparticules :
biovectors
Transport of albumin-loaded biovectors
Pe (x10-3 cm/min)
1
0,8
0,6
X 27
0,4
0,2
0
Albumin
Albumin-loaded biovectors