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An in vitro Monkey BBB Model Shinsuke Nakagawa1,2, Mária A. Deli2,3, Dinh Ha Duy Thuy2, Mayumi Sagara2,

Gohei So1, Narumi Yamada1, Rie Tatsumi1,2, Kunihiko Tanaka1, Masami Niwa1,2

1 Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, 2 BBB Laboratory, PharmaCo-Cell Company Ltd., 1-12-4 Sakamoto, Nagasaki 852-8523, Japan, 3 Institute of Biophysics, Biological Research Center of Hungarian Academy of Sciences, Szeged, Hungary

- - - -

+ + + + +

+

+ + + + +

+

+ + + + +

+

+ + + + +

+

3. Receptors

Enzymes

Metabolism

1. Transendothelial & junctional permeability

2. Transporters Efflux -Influx

Tight junction

Brain Capillary Endothelial Cells

4. Crosstalk

Astrocyte

Pericyte

Neuron

astrocytes

pericytes

endotheliall cells

BBB in brain capillary endothelial cells is

functionally maintaining in collaboration with

neighboring cells such as astrocytes, pericytes

and neurons, in the neurovascular unit.

Damages of BBB are closely related to on

onset and progress of neurological disorders.

Blood-Brain Barrier (BBB) mouse Cumbersome in handling with small size of materials Comparison with in vivo Data

Rat Cumbersome in handling with small size of materials Comparison with in vivo Data

Bovine Easy handling Difficult in Comparison with in vivo Data

Porcine Easy handling Difficult in Comparison with in vivo Data

Human The best in vitro BBB model Difficult in getting Cells

Immortalized cells Immatured Tight Junctions

Purpose

To elucidate the differences between rodent and primate BBB-related data, and

measure permeability of novel drugs which are under research and development

with human-related BBB, we need a in vitro monkey BBB model.

In vitro BBB

model

Macaca fascicularis (Macaca irus)

Primates Simiiformes Cercopithecinae Macaca

English name Crab-eating Macaque

Scientific Name Macaca irus

Distribution Southeast Asis

Living area Seaside, Riverside, wetland

Height 40～50cm

Tail 40～50cm

BW 5 kg

Life span 30 years

Monkey

3-year-old male 4

4-year-old female 1

13-year-old male 1

(For microvessels isolation) (For pericytes) astrocytes

2 g

Papain

Poly-L-lysin coated 75cm2 flask

5 g 35 g

collagenase CLS2 collagenase CLS2

subculture

2~3 weeks

20% BSA

Percoll

Coating(-) 100 mm dish (p0)

20% BSA

Percoll

Coating(+) 100 mm dish

collagenase /dispase

Subculture (p1) 1 week

subculture(p2)

Primary culture of monkey BBB-related cells

滅菌した濾紙に付着させることで硬膜、くも膜除去

Brain：42ｇ/one monkey

MBEC

vWF

ZO-1 occludin

claudin-5

ZO-1

occludin

claudin-5

Tight Junction

Target Proteins in Monkey Brain

Microvascular Endothelial Cells (MBEC)

Immunopositive expressions of von Willebrand factor,

Claudin-5, Occludin, and ZO-1

Result 1

day1 day3 day4 day5 0

150

250

200

day2 day6 day7

100

50 TE

ER

(Ω

x c

m2)

13 yr （1） 4 yr （1） 3 yr （4）

TEER of monolayer of MBEC increased up to over

100 Ω x cm2 at Day 3, including TEER of 13 yr old Male.

Result 2 TEER（Trans-endothelial

electrical resistance)

E00

R 123

P-gp

cyclosporin

(P-gp substrate)

**p < 0.01 vs. control (n=4), day 4

**p < 0.01 vs. control (n=3), day 8

B B

A A

Brain（B）

Blood（A）

BCRP

Monkey Rat

P-gp

- - - -

+++++

+

+++++

+

++++++

++++++

TransportersInflux -Efflux

Tight junction

GLUT1(d-glucose)

Pgp, MRP1(vinca alkaloids,

cyclosporin A)血管側

（luminal)

脳側（abluminal)

R 1

23 a

ccum

ula

tion

(nm

ol/

mg

pro

tein

)

1.0

0

0.5

1.5

**

control CsA (10μM)

R 1

23

Pe

(x

10

-6 c

m/s

)

5

**

15

0

10

20

A significant amount of functional P-gp was detected in

MBEC, with a dominant direction of brain- to blood-side.

Result 3 Transporter （P-glycoprotein, P-gp）

1. Barrier functions

2. Transpoters

3. Enzymes

TJs protein↑ TEER↑ Pe↓…

P-gp ↑ GLUT1↑…

γ-GTP ↑ ALP↑…

pericyte

astrocyte

endothelium

Cross-Talks between BBB-related Cells

Co-Culture Effects E00

EP0

E0A

EPA

day2 day3 day4 day5

E00

EP0

E0A

EPA

0

150

250

350

200

300

400

TE

ER

(Ω

x c

m2)

Transwell PET-12-0.4

monkey endothelial, rat pericytes, rat astrocytes

0

1

2

3

5

6

7

4

Na-

F P

e (

x 1

0-6

cm

/s)

E00 E0A EP0 EPA

** ** **

**p < 0.01 vs. control (n=3)

Result 4

Rat pericytes (P) and astrocytes (A) drove MBEC to have

the highest TEER (350 Ω x cm2 ) in co-culture BBB model

of EPA (BBB KitTM).

Co-culture BBB Model

EPA

E0A

EP0

E00

*p < 0.05 , **p < 0.01 vs. control (n=3)

claudin-5

β-actin

E00 E0A EPA EP0

claudin-5

CL

DN

5 /

β-a

ctin

(% o

f E

00

)

E00 E0A EP0 EPA

100

150

200

0

** *

*

Result 5

Rat pericytes (P) and astrocytes (A) collaboratively

Drove MBEC to the highest expression of claudin-5

in co-culture BBB model of EPA (BBB KitTM).

Co-culture BBB Model

MW CNS Transport

caffein 212 + passive lipophilic

antipyrine 188 + passive lipophilic

L-phenylalanine 165 + influx (L-system)

verapamil 491 ± efflux

cyclosporin A 1202 - efflux

digoxin 781 - efflux

sucrose 342 - passive hydrophilic

sodium fluorescein (Na-F) 376 - passive hydrophilic

R² = 0.9549

In vitro Monkey BBB model

caffein

antypirine

verapamil

Na-F sucrose

digoxin

In v

itro

Rat

BB

B m

od

el

0 10 20 30 40 50

0

10

20

30

40

50

10

0

10

1

Papp (x10-6 cm/s)

caffein

antypirine

verapamil

Na-F

sucrose

digoxin

L-phenylalanine

cyclosporinA

Papp (x10-6 cm/s)

Result 6 BBB Permeability of Drugs

A very good correlation between Papp of drugs was

obtained from Rat and Monkey BBB models (BBB Kit)

had immunopositive expressions of

von Willebrand factor, Claudin-5, Occludin, and ZO-1.

had TEERs of over 100 Ω x cm2 (monolayer, Day 3).

Had a significant amount of functional P-gp with a

dominant direction of brain- to blood-side.

Monkey brain microvascular

endothelial cells (MBEC )

Monkey BBB KitTM

Rat pericytes and astrocytes

collaboratively drove MBEC to the

highest expression of claudin-5 in

the co-culture BBB model of EPA

(Monkey BBB KitTM).

Rat pericytes (P) and astrocytes (A) drove

MBEC to have the highest TEER (350 Ω x cm2 )

in Monkey BBB KitTM.

A very good correlation between Papp of

drugs was obtained from Rat and Monkey

BBB KitTM.

Monkey BBB KitTM is a new and useful tool for

basic research and BBB permeability assays. In

collaboration with rat BBB KitTM, monkey BBB KitTM

would pave the way to elucidate the differences

between rodent and primate BBB-related data.

Conclusion

VA – volume of abluminal chamber (1.5cm3) A – membrane surface area (1.12cm2) [C]L – initial luminal tracer concentration (M) [C]A – abluminal tracer concentration (M) Δt – time of experiment (sec)

VA

A × ［C］L

Papp

(cm/s) ×

［C］A

Δt =

Transwell PC-12-3.0

drugs

BBB Transport of Drugs into Brain

sampling

Δt

(30 min)

EPA

Introduction & Methods

Result Ⅰ

Result Ⅱ Summary