M achine Perfusion Duration (m in) M achine Perfusion Duration...
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PHARMACOKINETIC APPLICATION OF NORMOTHERMIC PERFUSED EX VIVO PORCINE LIVERS Lianne Stevens 1,2 Jeroen Dubbeld 2 Jason Doppenberg 2 Steven Erpelinck 1 Catherijne Knibbe 3 Ian Alwayn 2 Evita van de Steeg 1 1 TNO, Microbiology & Systems Biology, The Netherlands 2 Leiden University Medical Centre, The Netherlands 3 Leiden Academic Centre Drug Research, The Netherlands E: [email protected] INTRODUCTION Current models to predict biliary excretion often fail due to species differences (rodent/dog) or due to differences in transporter expression in in vitro assays (e.g. sandwich cultured hepatocytes). Especially when drugs are subjective to enterohepatic circulation (EHC), this results difficulties to predict plasma profiles after oral and iv administration. Moreover, in case a compound is subjective to EHC, it is more prone to cause any drug-drug interaction and/or drug induced liver injury. AIM We aimed to set-up a pre-clinical model to study hepatic clearance, biliary excretion and the effect of drug-drug interaction on these processes by applying whole porcine liver on a pressure-controlled perfusion machine (Liver Assist). METHODS Porcine livers were made available from the slaughterhouse. After termination, the portal vein and hepatic artery were cannulated and flushed with saline and HTK solution. Bile duct was cannulated and liver was connected to the LiverAssist (figure 1A and B). Livers were perfused under oxygenated (95% O2, 5% CO2) conditions at 37°C. Perfusate composition is shown in Table 1. A bolus injection of the drug of interest was applied at the portal vein and samples were taken in time of the perfusate and the bile. Every hour bloodgas analysis was performed to measure the functional and metabolic 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 3. Plasma kinetics (A) and biliary excretion (B) of rosuvastatin and N-desmethyl rosuvastatin for 180 min after bolus injection of 3 mg rosuvastatin to ex vivo cannulated and perfused porcine livers in absence and presence of hepatic uptake and biliary excretion transporter inhibitor rifampicin (600 mg/h, continuous infusion). Figure 4. (A) Plasma clearance of indocyanine green (ICG) after a bolus injection and after 60 min a bolus injection + 25mg rifampicin showing delayed plasma clearance. (B) bilirubin concentration in the bile after a bolus + continuous infusion of 600 mg Rifampicin. 0 100 200 300 400 0 200 400 600 800 1000 Billirubin concontration measured in bile Time (min) Bilirubin (μmol/L) + rifampicin 0 50 100 150 0 50 100 150 %ICG plasma clearance Time after dosing (min) % ICG plasma ICG + rifampicin ICG bolus 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 perfused under oxygenated and normothermic conditions. • We have demonstrated the feasibility of NMP of porcine livers as a valuable tool to study hepatic 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 enzyme expression and activity. A B A B 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 is measured 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 0 100 200 300 400 500 0 500 1000 1500 2000 Portal flow Machine Perfusion Duration (min) Flow (mL/min) 0 100 200 300 400 500 0 50 100 150 Arterial flow Machine Perfusion Duration (min) Flow (mL/min) 0 100 200 300 400 500 6.5 7.0 7.5 8.0 pH Machine Perfusion Duration (hr) pH 0 100 200 300 400 500 0 50 100 150 Cumulative Bile Production Machine Perfusion Duration (min) Cumulative bile production (mL) 0 50 100 150 200 250 300 350 6.5 7.0 7.5 8.0 0.00 0.05 0.10 0.15 pH Machine Perfusion Duration (min) pH Delta pH Perfusate Bile Delta A B C D E F 0 100 200 300 400 0 200 400 2000 6000 10000 ALT & AST Machine Perfusion Duration (min) U/L AST ALT 0 100 200 300 400 500 0 20 40 60 80 100 Oxygen consumption Machine Perfusion Duration (min) O2 (kPa) Arterial Venous 0 100 200 300 400 500 0 5 10 15 CO2 production Machine Perfusion Duration (min) CO2 (kPa) Arterial Venous E F
Transcript of 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
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
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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
