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

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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

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

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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

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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

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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

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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 severalcytochrome P450 enzymes, endow this dynamic interfacewith metabolic activity

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Applications of BBB models in drug discovery and development

Targetidentification

Hitidentification

Leadidentification & optimization

Discovery Phase

Targetvalidation ofBBB-relatedmechanisms

In silico BBBpermeabilityassessment.Selection ofcompounds tobe run in cell basedassays

Optimization of BBBpermeability, metabolismand toxicological profile ofcompounds, using cell basedassays with graduallymore sophisticated protocols

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Development Phase

Candidate drugPre nomination

Concepttesting

Developmentfor launch

BBBmechanisticandtoxicologicalevaluations

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

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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

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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)

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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

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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

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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 studyingmechanistic 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 typicalexperimental 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)

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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

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Development of novel human BBB cell line hCMEC/D3

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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.

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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

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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

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Schematic diagram summarizingdevelopment 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.

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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

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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

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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

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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

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Preliminary studies done in our lab

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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

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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

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RT-PCR and Western Blot results

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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.

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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 1200

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Cumulative Transport of Paracellular Markers in hCMEC/D3 Cells

[C14]Mannitol[C14]Urea

Time (minutes)

Cum

ulat

ive

Subs

trate

Tra

nspo

rt (%

of D

onor

)

0 15 30 45 60 75 90 105 1200

10

20

30

40

50

60

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Cumulative Transport of Representative POT Substrates in hCMEC/D3 Cells

[H3]Histidine[H3]Valacyclovir

[H3]Glycylsarcosine

Time (minutes)

Cum

ulat

ive

Subs

trate

Tra

nspo

rt (%

of D

onor

)

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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

Man

nitol

Urea

Glyc

ylsar

cosin

e

Histidi

ne

Valacy

clovir

0

1

2

3

4

5

6

7

Pa

pp

( x

10-5

cm

2/s

ec)

Calculated permeability coefficients for various substrates across hCMEC/D3 cells.

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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.