«IN VITRO MODELS OF THE BLOOD-BRAIN BARRIER AND THEIR USE IN DRUG DEVELOPMENT» Alicante esi Meeting, Sept Pr Roméo Cecchelli

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1 «IN VITRO MODELS OF THE BLOOD-BRAIN BARRIER AND THEIR USE IN DRUG DEVELOPMENT» Alicante esi Meeting, Sept Pr Roméo Cecchelli

2 The ageing of the population gives to neurodegenrative diseases a frightening future because they relate to a population more and more important

3 But 95 % of new synthetised molecules do not reach the brain. WHY?

4 Ehrlich 1885 Goldmann 1913

5 The BBB was located in brain capillaries The lenght in Human is around 650 km Its surface area is around 12 m 2 The mean distance between 2 capillaries is around 40 microns

6 Brain capillaries are a complex structure

7 Brain capillaries Endothelial cells Astrocytes

8 Astrocytes Basal lamina Pericyte Endothelial cell

9 The Blood-Brain Barrier Cellular and Molecular Biology William M. Pardridge 1993

10 The Blood-Brain Barrier Cellular and Molecular Biology William M. Pardridge 1993

11 Blood BBB : a physical barrier Apical plasma membrane Tight junction claudins ZO-1 ZO-2 cingulin ZO-2 ZO-1 ZO-1 ZO-3 Actin ZO-1 occludin ZO-3 ZO-1 ZO-1 AF6 7H6 JAMs / Cadherins Cadherins / Adherens junction Brain

12 Peripheral capillary Endothelial Cell Biology N. Simionescu and M. Simionescu 1988

13 Peripheral capillary Endothelial Cell Biology N. Simionescu and M. Simionescu 1988

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15 blood BBB: PHYSICAL BARRIER tight junction endothelial cell brain Low transcellular transport No paracellula passage

16 BBB : a metabolic barrier Blood L-DOPA L-DOPA MAO Drug-metabolizing enzymes P-gp Dopamine Degradation Brain

17 BBB : A METABOLIC BARRIER GST UGT-1A6 Pericyte MAO-B Glutamyl aminopeptidase CYP 450 CYP 1A1 (rat) CYP 1B1 (human) CYP 2B1 (rat) CYP 2B6 (human) Endothelial cells

18 Blood Transport processes through a cerebral endothelium Specific transport (receptor or transporter) «fluid-phase» transcytosis Paracellula pathway ZO ZA Brain

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20 Different techniques used to study the BBB

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22 3 H/ 14 C-labelled drug (to measure BBB permeability) 14 C/ 3 H sucrose/inulin (to measure cerebrovascular volume

23 RESEARCH DEVELOPMENT BIRTH LIFE 1 to 2 years 1 to 2 years 1 to 2 years 6 to 8 years 1 to 2 years SEARCH FOR CHEMICAL STARTING POINTS Conception Synthesis SCREENING OPTIMISATION Potency Selectivity Oral bioavailability Duration of action IN VITRO & ANIMAL PRECLINICAL EVALUATION Activity Characterisation Stability Safety Efficacy in whole organism CLINICAL HUMAN EVALUATION PHASE I Tolerance & pharmacokinetics PHASE II Biological Activity & research of a therapeutic effect PHASE III Confirmation of safety & therapeutic effect FILE SUBMIS- SION/ REGIS- TRATION MARKETING APPROVAL PHASE IV

24 In vivo techniques These techniques can not be used in screening to evaluate the brain penetration of a molecule

25 In vitro methods

26 Cell lines All immortalized brain capillary endothelial cell lines (RBE4, MBEC line, TR-BBB ) do not expressed must of the tight junction proteins and are very leaky. They cannot be used to study transcellular transport. I just want to focus my presentation on primary and long term culture of brain capillary endothelial cells.

27 Classical Method Gray matter Enzymatic digestion + Centrifugation Microvessel endothelial cells and pericytes Capillaries Arterioles Venules Pericytes Primary culture of microvessel endothelial cells

28 Arterioles Gray matter Isolated microvessels Capillaries Mechanical homogenization + Capillaries Filtration Veinules Extracellular Matrix Pure capillary endothelial cells

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30 What about the Blood Brain Barrier marker?

31 Freeze fracture examination of endothelial cells Transendothelial electrical resistance = 400 Ohms.cm 2

32 Histochemichal detection of g-gt in brain capillaries

33 Gamma-GT expression Units / (mg prot.min) x x x x x x x x F VIII % positive cells Tight junctions MAO ACE gamma-gt Isolated capillaries P1 P 2 P3 P4 BBCE P 5 P6 P 7

34 Conclusion Consequently, endothelial cells in culture alone can not be considered as a relevant blood-brain barrier model

35 The BBB localisation Astrocytes Endothelial cell

36 The in vitro BBB model Blood Brain Endothelial cells Glial cells

37 Glial cell repartition Astrocytes Oligodendrocytes Microglia

38 What about the g GT activity?

39 Histochemichal detection of g-gt in brain capillaries endothelial cells

40 Statistical study of 2D-PAGE area from BCECs Co-culture Solo-culture Important variation : overexpressed in co-culture No statistical significant variation

41 Tight Junctions Blood Apical plasma membrane Tight junction claudins ZO-1 ZO-2 cingulin ZO-2 ZO-1 ZO-1 ZO-3 Actin ZO-3 ZO-1 ZO-1 occludin ZO-1 AF6 7H6 JAMs / Cadherins Cadherins / Adherens junction

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45 The transendothelial electrical resistance raises from 400 Ohms.cm 2 in soloculture to 800 Ohms.cm 2 in coculture

46 Isolated Isolated capillaries capillaries BBCE in in coculture BBCE BBCE in in solo solo culture culture P-gp detection by Western Blot analysis k Da

47 Analysis of OCTN2 and P-gp immunofluorescent staining in apical and basolateral membrane of endothelial cells OCTN2 P-gp nuclei Fluorescence intensity Slides (1 = 0.16μm) 24μm Apical membrane Basolateral membrane

48 Inhibition study (Uptake) VINCRISTINE %/control Control S9788 (1 M)

49 Conclusion In coculture with astrocytes, brain capillary endothelial cells present most of the characteristics that are known to have important functions in vivo

50 How can we use this model to predict the drug brain penetration?

51 General working process Refreshing glial cells medium (twice a week) Preparation of glial cells culture medium Defrosting glial cells vial Refreshing BCEC medium (every 2 days) Preparation of BCEC culture medium 60mm dishes coating Defrosting BCEC vial Filters coating Trypsinisation Seeding Transport experiment Analytics Raw data processing 3 weeks 12 days Glial Cells BCEC Co-culture Experiments

52 BBB Permeability studies T + 15 min T + 15 min Dosage

53 SUCROSE Pe = 0,24 x 10-3 cm/min clearance in L PSf = 14,38 L/min R 2 = 0, PSt = 0,95 L/min 100 R 2 = 0, time in min

54 CAFFEIN -3 Pe = 59,60 x 10 cm/min clearance in l PSf = 16,16 L/min R = 0,99 2 PSt = 15,18 L/min 2 R = 0, Time in min

55 Correlation between permeability values obtained in vivo with the in vivo techniques (Brain( perfusion and Oldendorf) and in vitro with the BBB model In BUI x MW In vivo Dopamine 4 Acetylsalicilic Sucrose 3 2 R = 0.93 Oldendorf s Technique Acid Phenytoin Nicotine Propranolol Hydrocortisone Urea In Pe x MW(cm/s) In vitro BBB Model Caffeine In Pe x MW(m g/mi n) In vivo R = 0.98 Cerebral Perfusion Manitol Sucrose Antipyrin Phenytoin Hydrocortisone Urea Diazepam In Pe x MW(cm/s) In vitro BBB Model Caffeine Propranolol

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57 In Silico / In vitro correlation Correlation between BBB cell culture permeability and in silico prediction R2 = 0.52 => R = 0.72 Q2 = 0.50 bbp03m21 Compounds measured by Stefan Lundquist and Mila Renftel Research DMPK, Södertälje. In silico model developed by Markus Haeberlein, Chemistry Dept, Södertälje

58 Reproducibility - 6 last months - 3 different clones (B1, PK, Lau1) between P4 et P7-4 different technicians Pe (sucrose) = 0,28 ± 0,12 (n=81) Astrazeneca (Sweden) Pe (sucrose) = 0,32± 0,18 (n=52)

59 Ranking Example of product Pe (x 10-3 cm/min) Brain penetration Caffeine Nicotine Diazepam Antipyrine DiPhenylHydantoine 7.37 Urea 2.22 Morphine 1.90 Verapamil 1.74 Warfarin 1.72 L Dopa 1.29 Vinblastine 1.19 Alanine 1.07 Leucine 0.91 Lactic acid 0.63 Sucrose 0.58 Glycerol 0.44 Cyclosporin 0.42 AZT 0.39 Cimetidine 0.26 Digoxin 0.21 Vincristine 0.15 Inulin 0.04 VERY GOOD GOOD LOW VERY LOW cm/min cm/min cm/min

60 This in vitro model constitutes a RELEVANT model to the BBB

61 RESEARCH DEVELOPMENT BIRTH LIFE 1 to 2 years 1 to 2 years 1 to 2 years 6 to 8 years 1 to 2 years SEARCH FOR CHEMICAL STARTING POINTS Conception Synthesis SCREENING OPTIMISATION Potency Selectivity Oral bioavailability Duration of action IN VITRO & ANIMAL PRECLINICAL EVALUATION Activity Characterisation Stability Safety Efficacy in whole organism CLINICAL HUMAN EVALUATION PHASE I Tolerance & pharmacokinetics PHASE II Biological Activity & research of a therapeutic effect PHASE III Confirmation of safety & therapeutic effect FILE SUBMIS- SION/ REGIS- TRATION MARKETING APPROVAL PHASE IV

62 General working process Refreshing glial cells medium (twice a week) Preparation of glial cells culture medium Defrosting glial cells vial Refreshing BCEC medium (every 2 days) Preparation of BCEC culture medium 60mm dishes coating Defrosting BCEC vial Filters coating Trypsinisation Seeding Transport experiment Analytics Raw data processing 3 weeks 12 days Glial Cells BCEC Co-culture Experiments

63 General working process Pack 1 day : C ELLIAL BBB-inducing medium Preparation of BCEC culture medium 60mm dishes coating Defrosting BCEC vial Filters coating Trypsinisation Seeding Transport experiment Analytics Raw data processing 4 days Preparation of in vitro BBB model Experiments

64 General working process Refreshing glial cells medium (twice a week) Preparation of glial cells culture medium Defrosting glial cells v ial Refreshing BCEC medium (every 2 days) CT Bovial@Screen Pack Preparation of BCEC culture medium 60mm dishes coating Defrosting BCEC v ial Filters coating Trypsinisation Seeding Transport experiment Analy tics Raw data processing 3 weeks 12 day s Glial Cells BCEC Co-culture Experiments 1 day : CELLIAL BBB-inducing mediu 4D@Screen Pack Transport experiment Analy tics Raw data processing 4 day s Preparation of in v itro BBB model Experiments

65 Culture Time CT Pack / 4D@Screen Pack 3 weeks (glial cell culture) Co-culture + 7 days (confluence)+ 5 days : BBB ready 60mm-culture dish 4 day-culture 4 days : BBB ready

66 BBB characteristics Cell monolayer morphology: Pack Vimentin Actin Bar = 25 μm

67 Tight junctions: Occludin BBB characteristics ZO-1 Pack Claudin-1 Claudin-5 Bar = 25 μm

68 Control of BBB integrity Sucrose permeability Pack Integrity threshold 12 day- Co-culture 4 dayculture

69 P-glycoprotein Pack Presence of the protein (Western Blot) Functionality of P-gp (inhibition assay) MW (kda)

70 P-gp Transporters (mrna) RT-PCR from co-cultures, capillaries and 4 day-endothelial cells coc C 4 day-e MRP1 MRP4 MRP5 MRP6 -actin coc C 4 day-e

71 Seeding CT Pack / 4D@Screen Pack 90 filters 12 day-system 6 well-filters 1 vial of endothelial cells 3 x 60mm-culture dishes 226 filters 4 day-system 24 well-filters

72 Correlation CT Pack / 4D@Screen Pack Correlation between in vitro BBB (24w-4d permeability) and in vivo BU (brain perfusion) 10 All parameters were normalized for molecular weight Ln[PexVMW] (ml/g/min) BU R 2 = Ln[PexVMW] (cm/s) in-v itro BBB

73 Ln[PexVMW] (ml/g/min) BU Correlation CT Bovial@Screen Pack / 4D@Screen Pack Correlation between in vivo BU (brain perfusion) and in vitro MDCK (Papp) All parameters were normalized for molecular weight R 2 = Ln[PexVMW] (cm/sec) MDCK

74 How to regulate, dose, improve the cerebral transport? By testing hits or leads on the in vitro model to have an experimental result of reference for permeability By testing optimized compounds ( affinity, selectivity ) on the in vitro model to check the increase in permeability and select the best canditates By testing selected compounds on in vivo model to check the in vivo cerebral penetration The in vitro model is an complementary tool which provide data to help for decisions It allows mechanistic studies on a cellular and a molecular level (Pgp- Specific transporters, pathological conditions i.e. stroke, inflammation)

75 Use of the transcytotic pathway Blood Low density lipoproteines (LDL) ZO ZA Brain

76 Conclusion ZO Lamp 1 Légèrement acide ZA

77 Nanoparticules : biovectors

78 Transport of albumin-loaded biovectors 1 Pe (x10-3 cm/min) 0,8 0,6 0,4 0,2 X 27 0 Albumin Albumin-loaded biovectors

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