m achine perfusion duration (m in) m achine perfusion duration...

PHARMACOKINETIC APPLICATION OF

NORMOTHERMIC PERFUSED EX VIVO

PORCINE LIVERS

Lianne Stevens1,2

Jeroen Dubbeld2

Jason Doppenberg2

Steven Erpelinck1

Catherijne Knibbe3

Ian Alwayn2

Evita van de Steeg1

1TNO, Microbiology & Systems Biology, The Netherlands2 Leiden University Medical Centre, The Netherlands3 Leiden Academic Centre Drug Research, The Netherlands

E: lianne.stevens@tno.nl

INTRODUCTION

Current models to predict biliary excretion oftenfail due to species differences (rodent/dog) ordue to differences in transporter expression inin vitro assays (e.g. sandwich culturedhepatocytes). Especially when drugs aresubjective to enterohepatic circulation (EHC),this results difficulties to predict plasma profilesafter oral and iv administration. Moreover, incase a compound is subjective to EHC, it is moreprone to cause any drug-drug interaction and/ordrug induced liver injury.

AIM

We aimed to set-up a pre-clinical model tostudy hepatic clearance, biliary excretion andthe effect of drug-drug interaction on theseprocesses by applying whole porcine liver on apressure-controlled perfusion machine (LiverAssist).

METHODS

Porcine livers were made available from theslaughterhouse. After termination, the portalvein and hepatic artery were cannulated andflushed with saline and HTK solution. Bile ductwas cannulated and liver was connected to theLiverAssist (figure 1A and B). Livers wereperfused under oxygenated (95% O2, 5% CO2)conditions at 37°C. Perfusate composition isshown in Table 1. A bolus injection of the drugof interest was applied at the portal vein andsamples were taken in time of the perfusateand the bile. Every hour bloodgas analysis wasperformed to measure the functional andmetabolic status of the liver.

CONCLUSIONS

Figure 1. (A) Schematic representation of the liver connected to the LiverAssist and (B) the LiverAssist set up with the porcine liver in the laboratory

FUTURE PERSPECTIVES

Figure 4.

G H

Figure 3. Plasma kinetics (A)and biliary excretion (B) ofrosuvastatin and N-desmethylrosuvastatin for 180 min afterbolus injection of 3 mgrosuvastatin to ex vivocannulated and perfusedporcine livers in absence andpresence of hepatic uptakeand biliary excretiontransporter inhibitorrifampicin (600 mg/h,continuous infusion).

Figure 4. (A) Plasmaclearance of indocyaninegreen (ICG) after a bolusinjection and after 60 min abolus injection + 25mgrifampicin showing delayedplasma clearance. (B)bilirubin concentration in thebile after a bolus +continuous infusion of 600mg Rifampicin.

0 1 0 0 2 0 0 3 0 0 4 0 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

B illiru b in c o n c o n tra t io n

m e a s u re d in b ile

T im e (m in )

Bil

iru

bin

(µ

mo

l/L

)

+ r ifa m p ic in

0 5 0 1 0 0 1 5 0

0

5 0

1 0 0

1 5 0

% IC G p la s m a c le a ra n c e

T im e a fte r d o s in g (m in )

% I

CG

pla

sm

a

IC G + r ifa m p ic inIC G b o lu s

Transporter interactions

Perfusate

Red blood cells 1L

Plasma 1L

Sodium bicarbonate 8,4% 30 mL

Calcium gluconate 10% 10 mL

A

B

Table 1. Perfusate composition of the perfused liver

Continuous infusion

Insulin 100U/hr

Epoprostenol 8 µg/h

Taurocholate 2%w/v / h

• The porcine liver was functional and metabolically active for at least 6h while being perfusedunder oxygenated and normothermic conditions.

• We have demonstrated the feasibility of NMP of porcine livers as a valuable tool to studyhepatic clearance and biliary excretion of rosuvastatin, which was used as a model drug.

Currently we are investigating the effect of longterm liver perfusion on transporter and enzymeexpression and activity.

A B

AB

Figure 2. Parameters shown during ex vivo porcine liver perfusion. (A) Livers are perfused via the Hepatic Artery (B) and the Portal Vein (C)during perfusion, perfusate pH is monitored and shows stable pH values. (D) livers showed to produce bile (E) pH of the perfusate and bile ismeasured to study the viability of the perfused liver (F) The livers showed to consume oxygen while perfused under normothermic conditions

RESULTS

Functional and metabolic parameters measured during normothermic perfusion

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

P o r ta l f lo w

M a c h in e P e rfu s io n D u ra tio n (m in )

Flo

w (

mL

/min

)

s tu d y 3

S tu d y 4

S tu d y 5

S tu d y 7

S tu d y 8

S tu d y 9

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

0

5 0

1 0 0

1 5 0

A rte r ia l f lo w

M a c h in e P e rfu s io n D u ra tio n (m in )

Flo

w (

mL

/min

)

S tu d y 2

S tu d y 4

S tu d y 5

S tu d y 8

S tu d y 9

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

6 .5

7 .0

7 .5

8 .0

p H

M a c h in e P e rfu s io n D u ra tio n (h r )

pH

S tu d y 3

S tu d y 4

S tu d y 5

s tu d y 9

S tu d y 8

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

0

5 0

1 0 0

1 5 0

C u m u la t iv e B ile P ro d u c t io n

M a c h in e P e rfu s io n D u ra tio n (m in )

Cu

mu

lati

ve

bil

e

pro

du

cti

on

(m

L)

S tu d y 3

S tu d y 4

S tu d y 5

S tu d y 8

S tu d y 9

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0

6 .5

7 .0

7 .5

8 .0

0 .0 0

0 .0 5

0 .1 0

0 .1 5

p H

M a c h in e P e rfu s io n D u ra t io n (m in )

pH

De

lta p

H

P e rfu s a te

B ile

D e lta

A B C D

E F

0 1 0 0 2 0 0 3 0 0 4 0 0

0

2 0 0

4 0 0

2 0 0 0

6 0 0 0

1 0 0 0 0

A L T & A S T

M a c h in e P e rfu s io n D u ra t io n (m in )

U/L

A S T

A LT

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

0

2 0

4 0

6 0

8 0

1 0 0

O x y g e n c o n s u m p tio n

M a c h in e P e rfu s io n D u ra t io n (m in )

O2

(k

Pa

)

A rte r ia l

V e n o u s

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

0

5

1 0

1 5

C O 2 p ro d u c t io n

M a c h in e P e rfu s io n D u ra t io n (m in )

CO

2 (

kP

a)

A rte r ia l

V e n o u s

E F