Characterization of novel human blood-brain barrier (hCMEC/d3) cell line


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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.

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Characterization of novel human blood-brain barrier (hCMEC/d3) cell line

  1. 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. 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. 3  Background information on blood-brain barrier (BBB) and its importance in development of CNS actives
  4. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 16  Development of novel human BBB cell line hCMEC/D3
  17. 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. 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:, and P. O. Couraud, Institut Cochin, Departement de Biologie cellulaire, 22 rue Mechain 75014 Paris, France. E-mail:
  19. 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. 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. 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. 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. 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. 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. 25  Preliminary studies done in our lab
  26. 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. 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. 28 RT-PCR and Western Blot results
  29. 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. 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. 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. 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.