In vitro Blood-Brain Barrier model by sjw10519

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									    In vitro Blood-Brain Barrier model
                                                                                                                                Pharmaceutical R&D

                                                                                                                                                                                                                                     A primary rat in vitro blood-brain barrier model
                                                                                                                                                                                                                                     to predict drug candidate entrance to the brain
                                                                                                                                                                                                                                                                                                                                                                               nicolas.perriere@vigicell.fr


 Blood-Brain Barrier: Complex and active interface between blood and brain

                                                                                                               The Blood-Brain Barrier:                                                                                                                                                                          Paracellular          Passive         Efflux                 Influx         Trancytosis
                                                                        Foot astrocyte                                                                                                                                                                                                                              route              diffusion    transporters          transporters      specific routes
                                                                                          Pericyte
                                                                                                                             • Maintains brain homeostasis
                                                                                                                                                                                                                                                                                                  BLOOD
                                                                                                                             • Controls all flux between blood and brain
                                                                                                                                                                                                                                                                                             Tight
                                                                                                                                                                                                                                                                                                           Brain
                                                                                                                             • Protects from xenobiotics                                                                                                                                   junction
                                                                   us




                                                                                                                                                                                                                                                                                                       endothelial cell
                                  le
                                uc
                               N




                                                                                                               Particular properties of BBB’s endothelial cells are
                                                                          Lumen                                conferred by the cerebral microenvironment:                                                                                                                                        BRAIN
                                                                                                  Tight                                                                                                                                                                                                          Hydrophilic             Lipophilic                                    Specific
                                                                                                junction                     • Expression of tight junctions                                                                                                                                                     compounds              compounds                                     substrates
                 Basal
                 lamina                                                                                                      • Only specific passage                                                                                                                                                                  Properties of BBB are a combination of
                                                                                   Endothelial cell                                                                                                                                                                                                     passive diffusion, active and facilitated transport (efflux and influx).
                                                                                                                             • Expression of polarized transporters




 VigiCell offers a physiological in vitro model that maintains these specific properties

              1            Similar to in vivo                                                                                                                                                                                                                                                 2        Quality & Reproducibility
     In vivo characteristics preserved using our in vitro standardized model                                                                                                                                                                                                                Standard animal

                    • Primary culture                                                                                                                                                                                                                                                             • Strain: Crl:OFA(SD) rat, male, 2 weeks old
                                                                                                                                                                                                                                     Brain endothelial cell                                       • Fresh primary cells
                    • Rat syngenic coculture                                                                                                                                                       BLOOD
                      (endothelial cells [RBECs] + glial cells)                                                                                                                                                                      Astrocytes                                             Protocol strategy
                                                                                                                                                                                                   BRAIN
                    • Freshly isolated cells                                                                                                                                                                                                                                                Preservation of brain endothelium properties
                                                                                                                                                                                                                                                                                                  • Soft enzymatic digestions
     Tight junction expression, localization and functionality                                                                                                                                                                                                                                    • Short amplification
                 Claudin-3                                                 Claudin-5                    Occludin                   ZO-1                                                                                                                                                           • Reduced culture time to avoid dedifferentiation

                                                                                                                                                                                                                                                                                            Standardized protocol
   in vivo




                                                                                                                                                                                                                      Paracellular permeability                                             Minimization of cell culture intra- and inter-variability
                                                                                                                                                                                                                         (Pe : 10-3 cm/min)
                                                                                                                                                                                                                                     fluorescein
                                                                                                                                                                                                                                                                                                  • Positive selection of brain endothelial cells with BBB phenotype
                                                                                                                                                                                                         sucrose         inulin                     FD4         FD40           FD70
                                                                                                                                                                                                          0.10           0.05          0.12         0.05        0.015          0.01               • Seeding on inserts at D4 with a defined cellular density
   in vitro




                                                                                                                                                                                                                                                                                                  • Permeability study realized at D10
                                                                                                                                                                                                                       Low permeability values
                                                                                                                                                                                                                          similar to in vivo
                                                                                                                                                                                                                                                                                            Internal control
                 Tight junction staining in freshly isolated brain capillaries and RBECs.
                                   Junctional localization in both cases                                                                                                                                                                                                                          • A fluorescent dye is coadministrated with the drug candidate in each
                                                                                                                                                                                                                                                                                                    BBB insert
   Transporter expression, polarization and functionality
                                                                                                                                                                                                                                                                                 Pe
                                                                                                                                                                                                                                                                                                                             Perm(Fluorescein)AtoB = 0.12 ± 0.02 10-3 cm/min (n=40)
                    P-gp                                                      Bcrp                           Oatp-2                                      400
                                                                                                                                                                                                         A to B w/o P-gp inhibitor
                                                                                                                                                                                                                                          Efflux ratio = 3.24
                                                                                                                                                                                                                                                                           (10-3 cm/min)

                                                                                                                                                                                                         B to A w/o P-gp inhibitor
                                                                                                                                                                                                                                                                           0.813 ± 0.056
                                                                                                                                                                                                         A to B w/ P-gp inhibitor
                                                                                                                                                                                                         B to A w/ P-gp inhibitor
      in vivo




                                                                                                                                                         300

                                                                                                                                                                                                                                                                                              3        Services : screening & customized studies
                                                                                                                                       Quantity (pmol)




                                                                                                                                                                                                                                                                           0.533 ± 0.060


                                                                                                                                                         200                                                                                                               0.409 ± 0.022



                                                                                                                                                                                                                                                                           0.251 ± 0.014
                                                                                                                                                                                                                                                                                              CELL CULTURE
                                                                                                                                                         100                                                                                                                                     • Production of 2 cultures per week (up to 96 inserts)
      in vitro




                                                                                                                                                          0                                                                                                                                   ADAPTABILITY
                                                                                                                                                               0                                         20         40         60      80              100         120
                                                                                                                                                                                                                             Time (minutes)                                                      • Shipment and protocol adapted to your needs
        Transporter staining in freshly isolated brain capillaries and                                                                                                                                   Polarized transport of Vinblastin                                                         (concentrations, controls, reproducibility, mechanistic studies…)
       RBECs. Membrane expression of transporters similar to in vivo                                                                                                                                                                                                                             • Access to mass spectrometry facility

                                                                                  *                                                                                                                                                                                                           VALIDATION
                                                                  150                                    *
                                  [Daunorubicin] (% of control)




                                                                                                                                                                   [Daunorubicin] (% of control)




                                                                                                                                                                                                   100
                 A to B                                           125
                                                                                                                     A to B vs B to A                                                                                                                                   B to A                   • Control of membrane integrity for each culture insert
                                                                                                              Daunorubicin transport in the                                                                                *
          Daunorubicin                                            100                                          presence or not of specific                                                         75
                                                                                                                                                                                                                                                                Daunorubicin                     • Control of P-gp functionality for each culture
           transport                                                                                           efflux transporter inhibitors                                                                                                                     transport
                                                                  75
                                                                                                                                                                                                   50

                                                                  50
                                                                                                                    Polarized transport                                                                                                                                                       OUR COMMITMENT
                                                                                                                                                                                                   25
                                                                  25                                                 of Daunorubicin                                                                                                                                                            • Permeability experiments within 8 days after reception of the drug
                                                                    0
                                                                          -     P-gp   Mrps      Bcrp
                                                                                                                                                                                                     0
                                                                                                                                                                                                               -         P-gp   Mrps      Bcrp                                                     candidate
                                                                                Transporter inhibited                                                                                                                    Transporter inhibited                                                  • Your analysis within 24h after assay results are received

                                                                                             Predictive pharmacokinetics
                                                                   in vivo        In vivo Kin
                                                                                    (µL/s/g)     Diffusion
                                                                                                                                                                                                                           R = 0.94                                                           4        Publications
                                                                                  100                                                                              Imipramine
                                                                                                 Glut-1influx                                                                                                                                                                                   • Perrière N et al. Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the
                                                                                                 P-gp efflux                                                                                                                  Diazepam                                                            expression of blood-brain barrier-specific properties. J Neurochem. 2005 Apr;93(2):279-89.
                                                                                   10            Paracellular route                                                                                                                                                                             • Perrière N et al. A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux
                                In situ Brain Perfusion *                                                                                                                                                                                     in vitro       brain
                                                                                                                                                                                                                                                                                                  transporters. Brain Res. 2007 May 30;1150:1-13.
                                                                                                 Combined passages              D-Glucose                                                                                                                  endothelial
                                                                                                                                                                                                              Prazosine                                       cells
                          In vivo technique which permits
                          to strictly measure passage of a
                                                                                      1                                                                                                                                                                                                         • Adenot M et al. Applications of a blood-brain barrier technology platform to predict CNS penetration of
                           chosen molecule through the
                                                                                                                Vinblastine                                                                                                                                astrocytes
                            BBB by radioactive detection                                                                                                  Colchicine                                                                       Syngenic co-culture of                                 various chemotherapeutic agents. 1. Anti-infective drugs. Chemotherapy. 2007;53(1):70-2.
                            without peripheral influences                                                                                                                                                                                  freshly isolated rat cells
                                                                                   0.1                Vincristine
                          *mice data from JM Scherrmann
                                                                                                                             M6G
                                                                                                                                                                                                                                                (astrocytes and
                                                                                                                                                                                                                                                 capillary brain
                                                                                                                                                                                                                                                                                                • Pifferi F et al. n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood-brain
                                                                                             Inulin                          Sucrose
                                                                                                                                                                                                                                               endothelial cells)                                 barrier.Prostaglandins Leukot Essent Fatty Acids. 2007 Nov-Dec;77(5-6):279-86.
                                                                                  0.01                                                                                                                                                          In vitro Pe
                                                                                                                                                                                                                                                  -3
                                                                                     0.01                              0.1                                                                           1                                     10 (10 cm/min)
                                                                                                                                                                                                                                                                                           nicolas.perriere@vigicell.fr
                    Strong correlation between in vitro and in vivo permeabilities, independent of the passage mechanisms                                                                                                                                                                  Tel. : +33 1 49 58 34 79
                                                                                                                                                                                                                                                                                           7 rue Guy Moquet 94800 Villejuif FRANCE


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                                                                                                                                                                                                                                     www.vigicell.fr & www.vigicell.eu

								
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