Characterization of novel human blood-brain barrier (hCMEC/d3) cell line for potential screening and uptake studies of pharmaceutical molecules with brain as their therapeutic target area. This is equivalent to Caco-2 cell lines routinely used for analysis of gastro-intestinal absorption of drugs.

1. Characterization of novel human blood-brain
barrier cell line (hCMEC/D3) for potential
screening of pharmaceutical molecules
- Debanjan Das
Ref: ABC and SLC Transporter Expression and Proton Oligopeptide Transporter (POT) Mediated Permeation across the
Human Blood–Brain Barrier Cell Line, hCMEC/D3” Debanjan Das et al. Mol. Pharmaceutics, 2012, 9 (12),
pp 3606–3606

2. 2
Outline
 Background information on blood-brain barrier
(BBB) and its importance in CNS drug
development
 Development of novel human BBB cell line
hCMEC/D3
 Preliminary characterization studies on
hCMEC/D3
 Effects of xenobiotic exposure on hCMEC/D3
 Future directions

3. 3
 Background information
on blood-brain barrier
(BBB) and its
importance in
development of CNS
actives

4. 4
 The market for neuropharmaceuticals is potentially one
of the largest sectors of the global pharmaceutical
market
 Many promising drug candidates fail due to the presence
of barriers between blood and brain present in cerebral
capillaries (BBB)
 Cerebral capillaries comprise approximately 95% of the
total area of the barriers between blood and brain
 BBB poses the main entry route for molecules into the
central nervous system (CNS)
 It impedes most neuropharmaceuticals from eliciting a
desired pharmacological effect at an attainable dose

5. 5
BBB – salient characteristics
 It has a total length of 650 km and a total surface
area of between 10–20 m2 of capillaries in the human
brain
 Complex tight junctions make the brain practically
inaccessible for polar molecules unless they are
transferred by transport pathways at the BBB that
regulate the microenvironment of the brain
 BBB is implicated in pathologies such as
neurodegenerative disorders, such as, Alzheimer’s
disease and multiple sclerosis), stroke and traumatic
brain injury, infectious processes and inflammatory pain

6. 6
 BBB dysfunction in these pathologies may result in
compromised transport and permeability
 This leads to alterations in cerebrovascular regulatory
mechanisms of blood flow, with ensuing abnormal
signaling between brain endothelium and associated
cells, such as glia and neurons
 By modeling BBB it is possible to make predictions about
brain uptake of potential drug candidates and to study
the effect of therapeutic interventions at the level of the
cerebral capillaries
 This provides not only powerful means to assess the risk
of taking compounds further in the pharmaceutical
development process but also generates important
information that allows for rational drug design

7. 7
Schematic
Representation
of BBB
Adapted from: Adapted from: Modelling of the blood-brain barrier in drug discovery and
development, Cecchelli R et al, Nat Rev Drug Discov. 2007 Aug;6(8):650-61
Intracellular and
extracellular
enzymes, such as
monoamine
oxidase (MAO), -
glutamyl
transpeptidase ( -
GT), alkaline
phosphatase,
Specific
peptidases,
nucleotidases and
several
cytochrome P450
enzymes, endow
this dynamic
interface
with metabolic
activity

8. 8
Applications of BBB models in drug discovery and
development
Target
identification
Hit
identification
Lead
identification
&
optimization
Discovery Phase
Target
validation of
BBB-related
mechanisms
In silico BBB
permeability
assessment.
Selection of
compounds to
be run in cell
based
assays
Optimization of BBB
permeability,
metabolism
and toxicological
profile of
compounds, using
cell based
assays with gradually
more sophisticated
protocols

9. 9
Development Phase
Candidate
drug
Pre
nomination
Concept
testing
Development
for launch
BBB
mechanistic
and
toxicological
evaluations
Cell models of the BBB, using different protocols, are used to evaluate
chemical modifications and feedback information to medicinal chemists
to allow optimization of properties governing brain uptake. In the
development phase, BBB cell models can also be used to address
specific aspects concerning, for example, mechanisms of action and
toxicology
Submission &
launch

10. 10
Transporters in BBB
 Brain endothelial cells contain numerous membrane
transporters on the luminal and abluminal membranes of
the capillaries that regulate the transcellular traffic of
essential molecules between brain and blood, as well as
effluxing potentially harmful substances and waste products
 Large molecules such as antibodies, lipoproteins, proteins
and peptides can also be transferred to the central
compartment by, for example, receptor-mediated
transcytosis or non-specific adsorptive-mediated
transcytosis
 Although the cerebral endothelium has a much lower
endocytotic/transcytotic activity compared with the
peripheral endothelium, it appears that these transport
mechanisms can be substantially up regulated at the BBB in
pathological conditions

11. 11
 BBB transporters exist for a variety of molecules, such as
amino acids, glucose, micronutrients, electrolytes,
hormones and peptides, and not all operate equally well
in both the blood-to-brain and brain-to-blood direction
 Of special interest for strategies to deliver drugs to the
CNS are the efflux transport systems: P-glycoprotein (P-
gp) and the multidrug resistance-associated protein
family (MRP)

12. 12
Criteria for BBB models
 Any drug discovery or development program involving
compounds targeted to the CNS needs to take the properties of
the BBB into account to achieve relevant CNS exposure, but it is
also beneficial to determine the BBB permeability of peripherally
acting drugs as CNS mediated side-effects are unlikely to occur
if permeability is low
 A well-characterized in vitro BBB cell model can also provide a
valuable tool for studying mechanistic aspects of transport as
well as biological and pathological processes related to the BBB
 To use any in vitro BBB cell model successfully it needs to fulfill
a number of criteria, such as reproducible permeability of
reference compounds, good screening capacity, the display of
complex tight junctions, adequate expression of BBB phenotypic
transporters and transcytotic activity
 In addition, the cell model should be reasonably robust and
display a physiologically relevant morphology

13. 13
Commonly used techniques
 Carotid artery single injection technique
 Microdialysis
 Autoradiography
 PET
 Intravital microscopy in combination with various staining
techniques
 Knock-out animals
 In vitro BBB models
 Possibility to assess permeability and involvement of
transporters and receptor mediated/adsorptive
transcytosis
 Can be used to estimate luminal to abluminal or
abluminal to luminal transport
 Cells from “knock out” animals can be used to establish
BBB models
 Relatively high throughput
 Suitable for optimizing BBB permeability
 Low noise level and easier to elucidate

14. 14
Modeling BBB in vitro
Glial soluble factors
secreted in culture medium
induce BBB phenotype in
the capillary endothelium.
This can be used for
compound screening in the
drug discovery process, for
studying
mechanistic aspects of
BBB transport & other
biological and pathological
processes.
Brain endothelial cells are
grown on filter inserts
together with glial cells at
the bottom of 6-, 12- or
24-well culture plates
Illustration of a typical
experimental design which
allows a co-culture of brain
endothelial cells and glial
cells
(Adapted from Nature Reviews- Drug Discovery and Neuroscience 7, 41-53, January 2006)

15. 15
Summary
 With a relevant BBB model, it is possible to evaluate
whether a compounds’ interaction with brain endothelium is
likely to compromise its functionality or is likely to reach
and interfere with glial cells
 Other aspects that can be investigated may involve BBB
metabolism, inhibition of endogenous transporters and
effects of sequestration
 Such data may enhance the value of the toxicological
results generated in animals, both in terms of
understanding the toxicity tests and in comparison with
clinical data, in the assessment of risk and safety in humans
 However, the screens that are currently available usually do
not allow high enough throughput to efficiently evaluate the
large number of compounds generated by pharmaceutical
and chemical companies

16. 16
 Development
of novel
human BBB
cell line
hCMEC/D3

17. 17
First stable human BBB cell line
 Although primary cultures of human brain endothelial
cells have been shown to retain some phenotypic
characteristics of brain endothelium, they rapidly
undergo dedifferentiation and senescence even upon
limited passaging, thus hampering usefulness as in
vitro models of the human BBB.
 Recently, transgenic expression of the catalytic unit of
telomerase (hTERT), alone or in combination with an
oncogene, has been shown to prevent telomere
shortening, to extend cellular lifespan and in some
cases to immortalize human endothelial cells of
different peripheral organs in culture.

18. 18
 An immortalized human brain endothelial cell line,
hCMEC/D3*, derived from a primary cell culture
through co-expression of hTERT and the SV40 large T
antigen via a highly efficient lentiviral vector system.
This cell line is claimed to retain most of the
morphological and functional characteristics of brain
endothelial cells, even without coculture with glial
cells and may thus constitute a reliable in vitro model
of the human BBB
*Ref: Blood-brain barrier-specific properties of a human
adult brain endothelial cell line** – Weksler and
Couraud; **The FASEB Journal express article 10.1096/fj.04-
3458fje. Published online September 1, 2005. Corresponding
authors: B. B. Weksler, Weill Medical College of Cornell University,
New York, NY, 10021, USA. E-mail: babette@mail.med.cornell.edu,
and P. O. Couraud, Institut Cochin, Departement de Biologie
cellulaire, 22 rue Mechain 75014 Paris, France. E-mail:
couraud@cochin.inserm.fr

19. 19
Morphological characteristics of hCMEC/D3 cells
Phase contrast microscopic view of the primary
culture of human brain endothelial cells showing
elongated, tightly packed, contact inhibited
morphology (A) and of the immortalized hCMEC/D3
clonal cell line (B), with a similar morphology

20. 20
Schematic diagram summarizing
development of cell line and its properties
Adapted from: Blood-brain barrier-specific properties of a human adult brain endothelial cell line,
Weksler et al, The FASEB Journal. 2005;19:1872-1874.

21. 21
Permeability Studies
Permeability assays using fluorescent dextrans of increasing
molecular size (4–70 kDa) revealed that hCMEC/D3 monolayers
exert a better restriction than primary cultures of bovine brain
endothelial cells and a much more stringent restriction than do
GPNT cells on the trans endothelial passage of both low
molecular-weight and high-molecular-weight dextran molecules

22. 22
 With reference bovine BBB coculture model, the permeability
coefficient for [14C]-sucrose (MW=340 Da) was higher for
hCMEC/D3 cells (1.65 vs. 0.75×10 −3 cm/min), but in the
case of [3H]-inulin (MW=4,000 Da), the permeability for both
models was of the same magnitude (0.36 vs. 0.37×10−3
cm/min
 Transendothelial electric resistance resistance (TEER) was
found to remain constantly at low levels (<40 Ω.cm2),
reflecting a high ionic permeability

23. 23
 A) Correlation between in vitro
permeability of hCMEC/D3 cells
and reported in vivo BBB
permeability for a variety of
chemical compounds. In vitro
permeability of indicated drugs
across confluent monolayers of
hCMEC/D3 cells on polycarbonate
Transwell filters is expressed as
permeability coefficients (Pe, 10-3
cm/min)
 Results of in vivo BBB permeability
for the same compounds
(expressed as transport
coefficients: Kin, 10-3 ml.s-1.g-1 )
were assessed by the brain
perfusion technique in adult rats
or mice
 Tested compounds are [14C]-
diazepam and -morphine-6-
glucuronide (M6G), [3H]-
imipramine, -prazosin, -colchicine
and -vincristine
 Correlations between the in vitro
permeability data for [14C]-
diazepam, [3H]-prazosin, -
colchicine and -vincristine
obtained with hCMEC/D3 cells (y-
axis) and rat brain GPNT cells
 (B) or with hCMEC/D3 cells (y-
axis) and HUVECs
 (C) are presented for comparison

24. 24
Summary
 The hCMEC/D3 cell line is the first example of
an extensively characterized human brain
endothelial cell line that expresses most of the
unique properties of the BBB, even without
coculture with glial cells
 This cell line demands immediate attention for
further characterization and optimization for
widespread application

25. 25
 Preliminary
studies done in
our lab

26. 26
Expression and transport kinetics study
due to various Proton Oligopeptide
Transporters
 To assess the validity of using this cell line to model the expression and
transport kinetics due to various POT-members the following methodologies
are used:
 The hCMEC/D3 cell line was maintained in a modified EGM-2 medium
(Lonza, Walkersville, MD) in collagenated culture flasks and passaged every
3-4 days at approximately 85%-95% confluence
 Total protein and RNA were extracted at passages 5, 8, 12, 15, and 20 and
respective protein and mRNA expressions for POT
members, (PepT1, PepT2, PHT1 and PHT2) were determined by Western
blotting and RT-PCR comparative to β-actin
 Transport studies were conducted using [H3]- histidine, [H3]-
glycylsarcosine and [H3]- valaciclovir in collagen-coated 6 well Transwells
 The integrity of the hCMEC/D3 monolayer was evaluated by transepithelial
electrical resistance (TEER) and [C14]- urea and [C14]- mannitol transport

27. 27
POT expression studies
Bright field images of
hCMEC/D3 cells seeded at
A) 1.0 x 105 cells/cm2; B)
1.5 x 105 cells/cm2; and C)
2.0 x 105 cells/cm2. Images
were acquired on an
Olympus BX-51 light
microscope at 10x
magnification.
a
b
c

28. 28
RT-PCR and Western Blot results

29. 29
Permeability Studies
 Calculation of Effective Pore Radius:
(1)
(2)
(1)Assuming a single pore model, the dimensionless Renken
molecular sieving function compares the molecular radius (r) and
the cylindrical pore radius (R) and takes values of 0 < F(r/R) <
1.
(2)The aqueous pore radius was calculated from (2) using the ratio
of the paracellular permeabilities of [C14]- Mannitol and [C14]-
urea.

30. 30
POT mediated transport
Transport kinetics across hCMEC/D3 cells. Cells were seeded at 2 x 105
cells/cm2 on 24 mm PVDF Transwells.
A) Transport of passive paracellular markers ([C14]- mannitol and
[C14]- urea) were used to calculate the effective intercellular pore
radius per (2) above.
B) Transport of representative POT substrates: [H3]- glycylsarcosine
for PepT1 mediated transport, [H3]- histidine for PHT1-mediated
transport and [H3]- valacyclovir representing mixed effect transport.
0 15 30 45 60 75 90 105 120
0
10
20
30
40
50
60
70
80
90
100
Cumulative Transport of Paracellular Markers inhCMEC/D3 Cells
[C14
]Mannitol
[C14
]Urea
Time (minutes)
CumulativeSubstrateTransport(%ofDonor)
0 15 30 45 60 75 90 105 120
0
10
20
30
40
50
60
70
80
90
100
Cumulative Transport of Representative POT Substrates
inhCMEC/D3 Cells
[H3
]Histidine
[H3
]Valacyclovir
[H3
]Glycylsarcosine
Time (minutes)
CumulativeSubstrateTransport(%ofDonor)

31. 31
Calculation of Permeability Coefficients
Compound Papp (x 10-5
cm/sec)
[C14]- Mannitol 2.47 0.02
[C14]- Urea 5.65 0.08
[H3]- Glycylsarcosine 4.20 0.17
[H3]- Histidine 4.41 0.17
[H3]- Valacyclovir 7.01 0.13
Calculated Permeability Coefficients for Various
Substrates Across hCMEC/D3 Cells
M
annitol
Urea
G
lycylsarcosine
Histidine
Valacyclovir
0
1
2
3
4
5
6
7
Papp(x10-5
cm2
/sec)
Calculated permeability coefficients for various substrates
across hCMEC/D3 cells.

32. 32
Conclusions
 hCMEC/D3 cells demonstrate stable expression of both
PHT1 and PHT2 with respect to passage number and days
post-seeding as determined by RT-PCR and Western
Blotting
 hCMEC/D3 cells do not express either PepT1, or PepT2 by
RT-PCR, which is consistent with the human BBB in vivo.
 [C14]- Mannitol and [C14]- urea transport studies and
observed TEER values indicate hCMEC/D3 cells form a
confluent monolayer that exhibits a pore radius of 19.39Å ±
0.84Å, as calculated using the single pore Renkin molecular
sieving function
 Although methods to increase the tightness are necessary
to demonstrate broader utility of the hCMEC/D3 cell line, it
does provide a surrogate model for studying human BBB
function.