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Laboratoire de Physiopathologie de la Barrière Hémato-Encéphalique EA 2465, IMPRT: IFR 114 Faculté des Sciences Jean Perrin Cellial technologies 62307 Lens- FRANCE OVERVIEW OF THE BLOOD-BRAIN BARRIER AND ITS PROPERTIES Pr Romeo CECCHELLI The Cerebral Vasculature and Neuroimaging in CNS Drug Dicovery And Development: Physiology,Pathology and Methodology Astrazeneca june 2006 Ehrlich 1885 Goldmann 1913 Peptide Drug Delivery to the Brain William M. Pardridge 1991 The classical view of the BBB formulated by this pioneering work was based on evidence that blood-borne substances were excluded from the brain The BBB was located in brain capillaries and considered as a rigid structure The Blood-Brain Barrier Cellular and Molecular Biology William M. Pardridge 1993 Peptide Drug Delivery to the Brain William M. Pardridge 1991 • Brain capillaries are a complex structure The Blood-Brain Barrier Cellular and Molecular Biology William M. Pardridge 1993 Where is located the barrier? Brain capillary endothelial cells are the physical components of the BBB Peptide Drug Delivery to the Brain William M. Pardridge 1991 The Blood-Brain Barrier Cellular and Molecular Biology William M. Pardridge 1993 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-3 ZO-2 ZO-1 occludin ZO-3 AF6 ZO-1 ZO-1 JAMs / 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 paracellular 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 Paracellular pathway ZO ZA Brain EXHAUSTIVE LIST OF BBB TRANSPORTERS from Terasaki T 2003 ? Different techniques used to study the BBB 3H/14C-labelled drug (to measure BBB permeability) 14C/3H –sucrose/inulin (to measure cerebrovascular volume BBB model IN VITRO BBB MODEL blood endothelial cells brain glial cells TRANSPORTERS AT THE BBB NUTRIENTS GLUT 1 ORGANIC ANIONS OAT4? MCT 1 OATP A oatp 2 ORGANIC CATIONS P-gp BCRP ATP Na+ A, B0,+ ASC OAT3 OCT? OCTN2 D D D D D P-gp protein (Confocal) - BBCEC P-gp OCTN2 nuclei Fluorescence intensity 30 20 10 0 0 5 10 15 Slides (1 = 0.16μm) Luminal membrane Abluminal membrane 24μm Developing new technology is necessary for progression of the BBB research 100 0 siRNA E2 (-) G 203 NC 50 E2 (+) 250 200 150 100 50 0 siRNA E2 (-) E2 (+) 300 250 200 150 100 50 0 siRNA E2 (-) G 203 NC 150 * G 203 NC 200 ABCC1/MRP1 ABCC1 mRNA (% of control) 250 300 G 203 NC * ABCB1/MDR1 G 203 NC 300 ABCB1 mRNA (% of control) ABCG2 G 203 NC ABCG2 mRNA (% of control) There is no selective inhibitors for distinguishing ABCC subtypes and ABCG2. Alternative strategy: siRNA siRNA can selectively suppress target transporter(s). E2 (+) G2-03, siRNA for ABCG2; NC, negative control siRNA Hori et al. J. Neurochem. 93:63-71 (2005) G2-03 siRNA selectively suppressed the expression of rat ABCG2 in brain capillary endothelial cells. Regulation of A in the CNS Brain Ab homeostasis is controlled by numerous pathways (Berislav,2005): (1) Peripheral and central production (2) Rapid receptor-mediated transport of soluble forms across the BBB from blood to brain via RAGE [30] (3) Similar transport across the BBB from brain to blood [41,43] via LRP [21,45] (4) Binding to transport proteins such as apoE, apoJ and a2-macroglobulin (a2M), which can influence: Ab sequestration in plasma, brain ISF and C SF; the form of Ab accumulation in brain (i.e. soluble versus fibrillar) [6,46,47]; and/or transport across the BBB and blood–C SF barrier [6,65] (5) Degradation, mediated by proteins such as enkephalinase [48], insulinase, plasmin, tissue plasminogen activator or matrix metalloproteinases [49], or mediated by astrocytes [50,51] and microglia [66] (6) Slow removal via ISF–C SF bulk flow [57] (7) Oligomerization and aggregation [5,6] Interaction between t-PA with the blood-brain barrier tPA and the Fibrinolytic System release tPA PAI-1 tPA pln activation inhibition 2-AP inhibition tPA plg pln Fibrin clot Breakdown FDPs Benchenane et al., Trends Neurosci 2004 Therapies Thrombolysis tPA Actilyse® However, increasing evidence supports the idea that t-PA could also potentiate stroke damage Does tPA cross the intact BBB in vivo? Collagen IV GFAP Intravenous Injection of Biotinylated Albumin Collagen IV Biot-albumin Albumin remains intravascular Intravenous Injection of Biotinylated Albumin or Biotinylated tPA Biotin Collagen IV Merged Albumin tPA tPA crosses the intact BBB in vivo Abluminal 15 tPA Abluminal tPA activity Luminal -loadedtPA tPA ctrl 5,0 (% ofloadedtPA) Man ZO-1Luminal Abluminal + 4,0 ? 3,0 2,0 tPA 1,0 pe 10 -3 cm/min (sucrose) Time (min) 30 60 + + 20 120 8 6 - rtPA + In vitro model of BBB 4 Tight junctions High transendothelial resitivity 2 0 Cecchelli et al., 1999 0,0 0 120 40 60 Ctrl 80 tPA 100 120 140 (min) tPA does not affect BBBtime integrity or permeability tPA crosses the BBB in vitro Mannitol How does tPA cross the BBB ? Transcellular Receptor-mediated Non specific Blood-brain barrier Paracellular Abluminal tPA activity (% of control) 140 120 100 80 60 ** 40 Biot-tPA (green) 20 0 37°C 4°C tPA crosses the BBB by transendothelial pathway Identification of the receptor involved in the passage of tPA Ctrl RAP Man 140 120 (% of control) Abluminal tPA activity tPA 100 80 60 40 * 20 0 Control RAP Mannose tPA crosses the intact BBB by LRP-mediated transcytosis There are a lot a sophiticated receptor-mediated transports at the BBB. Tomorrow, I will presenetd you the cellular mechanisms of these transports in physiological conditions and the influence of the surrounding cells on these transports The simple observation that epithelial but not endothelial cels are able to form a high-resistance and lowpermeability barrier in vitro sheds light on the importance of the microenvironnement in the maintenance of the barrier propeties in vivo Peptide Drug Delivery to the Brain William M. Pardridge 1991 Endothelial cells Astrocytes Statistical study of 2D-PAGE area from BCECs Co-culture Solo-culture Important variation : overexpressed in co-culture No statistical significant variation kDa 205 140 I ca sola pi te lla d rie s BB co C cu E i ltu n re BB CE cu in ltu so re lo P-gp detection by Western Blot analysis MRP6 MRP5 MRP1 MRP4 MRP6 MRP5 MRP1? MRP6 MRP4 MRP5 MRP4 MRP1? P-gp The definition of the BBB has shift today to a more integrated concept that takes into acount not only the bidirectionality of the exchange process, but also the discovery that besides the endothelium additional components (astrocytes, neurons) constitute integral parts of the barrier physiology Working for the BBB… Introduction HOW TO CROSS A CONTINUOUS CAPILLARY? Rippe et al., 2002 4 3.Transcytosis 4.Transporters 3 2 1 2. Transendothelial channel 1. Interendothelial clefts HOW TO REACH THE BRAIN? 1. Not between cells because of tight junctions 2. Not by transendothelial channel because of its lack 3 & 4. By transcellular processes Histochemichal detection of -GT in brain capillaries endothelial cells P-gp protein (Confocal) - BBCEC P-gp OCTN2 nuclei Fluorescence intensity 30 20 10 0 0 5 10 15 Slides (1 = 0.16μm) Luminal membrane Abluminal membrane 24μm CEREBRAL ENDOTHELIUM : METABOLIC AND TRANSPORT BARRIERS Gamma-GT Alcaline Phosphatase P-glycoprotein L-DOPA L-DOPA L-DOPA DCCA Glucose Dopamine Dopamine MAO-B DOPAC Leucine Na+ water Na+ Glycine Na+ K+ ATPase water Na+ Glutamate Introduction BBB CEREBRAL VICINITY cerebral capillary endothelial cells Pericytes Brain parenchyma cells Astrocytes Ctrl Ctrl Time Time (min) (min) 15 15 OGD OGD 30 30 60 15 60 15 30 30 60 60 (% of tPA loaded in the luminal side) tPA Activity in the abluminal side Abluminal Abluminal tPA tPA tPA 40 ** 30 Luminal ** OGD Control Abluminal 20 ? OGD 4h 10 Ctrl 0 0 15 30 60 Time (min) OGD potentiates the passage of tPA OGD Biot-tPA (red)
