BrainPlotting poster at the SEISC congress, december 2018

1. Our core business is the study of pharmacokine5cs (PK) in the Human brain at early
preclinical stage by using the most accurate predic5ve models and most recent
tools. To that end, we have developed a proprietary method: the Brain Exposure
Predic5on model (BEP) based on primary Human in vitro tools from fresh Human
brain 5ssues.
The BEP model provides the in vivo 5me-curves of drug free concentra5on in the
brain, based on in vitro generated PK brain parameters and PBPK modeling.
Brain Exposure Predic1on Func1onality of the BBB model
2. Equilibrium dialysis methods
Fresh Human brain slices
Brain 5ssue is alive and permits to highlight specific transport
mechanisms in neural cells.
We measure (fu) in ECF and ICF to determine drug concentra5ons
within the brain
Plasma & Brain Homogenates
We use Human plasma and brain 5ssues to measure binding to
Human lipids and proteins to improve predictability
Brain extracellular space
Vecf, Cecf, fu,brain ecf
Brain intravascular space
Viv, Civ, fu,plasma
Plasma compartment
Vpl, Cpl, fu,plasma
Dose
Qbr Qbr
CLp
CLinin
CLoutoutoutou
fu,brain icf , fu,brain ecf and fu,plasma : unbound
frac5on into the brain intra and
extracellular fluid and plasma
Viv (mL/kg): physiological volume of the
extravascular space
Vicf and Vecf (mL/kg): physiological
volume of the intra and extra-vascular
space
CL (mL/h/kg): clearances
Qbr (mL/h/kg): brain perfusion blood
flow
Vpl (mL/kg): volume of distribu5on of the
molecule into the plasma compartment
C (ng/mL): concentra5on of the
molecule
Brain intracellular space
Vicf, Cicf, fu,brain icf
BBB
We compute in vitro
human data to predict
Human in vivo PK
informa5on.
All results are obtained
from physiological
models
3. PBPK Modelisa1on (Physiologically Based PK)
Quan5fica5on of protein levels by a LC-MS/MS technique confirms the good
expression of 5ght junc5on proteins and ABC transporters (1).
Predic7on of Human PK data based on innova7ve
Human primary in vitro models
Focus on BrainPlo=ng's BBB model func7onality Human data from preclinical stages
Brain/Plasma
unbound frac5on
defined for your product
Conclusions
With physiological in vitro BBB models as close as possible to the in vivo
situa5on, we get op5mal permeability coefficients (Pe) and efflux ra5o (ER).
Tightness of the BBB model is evaluated by fluorescein permeability and Trans
Endothelial Electrical Resistance (TEER) which show a great 5ghtness result of
615 ± 304 Ω.cm2.
1. BBB model
(1) D. Gomez, M. Taghi, M.C. Menet ; INSERM UMR-S 1144 Faculté de Pharmacie Paris Descartes
(2) A. Moreau, C. Denizot, B. Walther ; Technologie Servier, Orléans
(3) S. Cisternino ; INSERM UMR-S 1144 Faculté de Pharmacie Paris Descartes
(4) H. Luo, X. Decleves ; INSERM UMR-S 1144 Faculté de Pharmacie Paris Descartes
BEP model predicts Human brain PK from in vitro derived parameters: the Pe and efflux
ra5o determined across the BBB and the drug unbound frac5on in plasma (fu,plasma) and
in brain 5ssue (fu,brain) determined by the equilibrium dialysis method, with brain
homogenates and brain slices.
Most of drugs are known to be
substrates of efflux ABC-transporters
like P-gp, Bcrp and the Mrps familly.
Their specificity and func5onality are
very species dependent.
We show with a few molecules (in red
on the chart) the presence and the
func5onality of such transporters which
result in a polariza5on of the cells. This
efflux ra5o is near to 2, which is close to
measured in vivo data.
This evalua5on was done in partnership
with Technologie Servier (2).
To reach its target, the
molecule of interest
has to cross the BBB
and get into the
extracellular (ECF) or
intracellular fluids (ICF)
of neural cells
Endocytosis
or Exocytosis
Facilitated
transport
Efflux
Simple
diffusion
Paracellular
pathway Tight
junc5ons BBB
Blood Stream
Brain Parenchyma
fu,plasma
fu,ecf
Brain Extracellular Fluid
(ECF)
fu,icf
Glial & Neuronal cells
Brain Intracellular Fluid (ICF)
We have developed in vitro models of the Human BBB based on Human fresh resec5ons. We obtained relevant reliable tools for PK screening of one or several
compounds, as well as specific studies which required a totally func5onal model.
French academic Laboratories of excellence, Big Pharma as well as small companies have already trusted to work with us.
• For each compound we are able to offer predic5ons for the concentra5on of the free drug in the brain parenchyma and more
par5cularly in the ICF and ECF.
• To that end we use exclusively physiological in vitro tools, developed at BrainPlolng, based on Human fresh resec5ons
• Depending on yours needs, we can also propose models, from brains affected with different pathologies with different
characteris5cs.
glial cell condi5oned
medium (h,i,j,k).
They are then
suitable for further
studies (d,e,f,g).
a b c
j
GFAP SMA (g)/GFAP (r)
k
Isolated Human brain capillaries
are seeded (a). Brain endothelial
cells sprout (b), are posi5vely
selected,shortly amplified (c),
and seeded on insert culture with
ZO-1
d
Claudin-5
e
Occludin
f
P-gp
g
h i
0,0
0,5
1,0
1,5
2,0
2,5
3,0
0,0 0,5 1,0 1,5 2,0 2,5 3,0
Clearance A to B (µl.min-1)
Clearance B to A (µl.min-1)
No Efflux Drugs
Efflux Drugs
Imipramin
Quinidin
Sulfasalazin
Ketoprofen
Verapamil
Diazepam
Paracetamol
Ofloxacin
Warfarin
Terbutalin
Carbamazepin
Propanolol
Indomethacin
4 - Digoxin
5 - Indinavir
Prazosin 1- Methotrexate
2 - CimeLdin
3 - Ciprofloxacin
AZT
0
2
4
6
8
10
12
Good ? Bad
Drugs number
Predic5on
Efflux
Ra5o 1
Efflux
Ra5o 2
1
2 3
4
5
Isola5on of capillaries
from fresh Human
brain resec5ons and
culture of brain
endothelial cells Vimen5n
Condi5oned Medium of
primary glial cells
“BLOOD: A”
“BRAIN: B”
Brain endothelial cells
2. Drug Transport at the BBB
1. Protein quan1fica1on of specific BBB proteins
Junc5on Proteins BBB Transporter
Type Culture n Claudin-5 PECAM-1 BCRP P-gp
Human brain
microvessels
Freshly extracted 4 ++ ++ ++ ++
Primary Human
BBB
BrainPlolng®
P1
4 ++ +++ + +
hCMEC/D3® On plas5c, P32 3 BQL + BQL ++
Proteomic expression:
BQL: Below QuanLficaLon Level,
+: low expression,
++: moderate expression,
+++: high expression.
Primary human endothelial cells and
microvessels extracted from resecLons
of gliomas,
hCMEC/D3 #SCC066 – Merck Millipore
Paracellular pathway
Small hydrophilic compounds have to cross the BBB through the 5ght junc5ons.
Fluorescein - 332 Da is used to that end. We observe non polarized very low permeability:
Fluorescein Pe AB = 0,13 ± 0,07 & Pe BA = 0,14 ± 0,07 x10-3 cm/min (n=30, n=14)
Transcellular pathway
Non efflux lipophilic compounds like imipramin, diazepam or propranolol cross the BBB
through the cell whose membrane is very specific (lipids composi5on and rates). We observe
non polarized high permeability (cf graphic below) (2).
Efflux ABC-transporters
SLC transporters
A lot of SLC transporters are present at the BBB. They are considered as facilita5ve transporters
able to transport their substrates with the concentra5on gradient. They are responsible for
example for D-glucose penetra5on in the brain and for transport of ions.
Func5onal proper5es are directly evaluated with the assessment of permeability.
0
50
100
A to B B to A
%
Phenylalanine
0
50
100
A to B
%
D-Glucose
0
50
100
A to B B to A
%
MPP+
Control
With Inhibitor
or Compe5tor
Genomic expression of Na+ coupled transporters
Type Culture n NHE1 NHE2 NHE3 NHE4 NHE5 NBCn1 NBCn2 NBCe1 NBCe2 NKCC1
Primary Human BBB BrainPlolng® P1 1 64 0,15 0,08 0,06 4,5 2,4 0,04 0,1 0,2 37
hCMEC/D3® On plas5c, P27 to P32 3 19 0,02 0,05 BQL 2,2 0,7 0,01 0,005 0,05 12
To demonstrate the presence
of SLC transporters, different
kinds of molecules are used.
Their transporta5on is studied
in the presence of inhibitors &
compe5tors, from A to B and B
to A (3).
Transcriptomic studies are also conducted to study the transport of ions (4):
Data expressed as raLo of each Na+-coupled transporter mRNAs compared with relaLve TBP mRNA expression (BQL: Below
QuanLficaLon Level). Primary human endothelial cells extracted from a resecLon of glioblastoma.