#3P-153

Lianne Stevens1,2
Joost Westerhout1
Jeroen Dubbeld2
Joanne Donkers1
Catherijne Knibbe3
Ian Alwayn2
Kazuhiro Ariga4
Evita van de Steeg1

OPTIMIZED NORMOTHERMIC MACHINEPERFUSION OF LIVER AND KIDNEYUSED TO PREDICT HUMAN
PHARMACOKINETICS

1 TNO, Human Cell Biology, Healthy Living, The Netherlands
2 LUMC, Transplantation Surgery, the Netherlands

3 LACDR, Systems Biomedicine and Pharmacology, The Netherlands
4 TNO Pharma, JapanRESULTS

INTRODUCTION
The prediction of hepatic clearance and biliary excretion is of highimportance to assess the pharmacokinetics of drugs.
Normothermic machine perfusion (NMP) of organs is a promising
tool to predict ADME of drugs, especially when combined withphysiologically-based pharmacokinetic (PBPK) modeling.

1) Solely taurocholate infusion during NMPACBAIMPerfusate delta
AST

403020100-1010000

Taurocholate

Bile acids

6000
4000
2000

0200

400600

Bile acid mix (n=2)
Blanco perfusion (n=3)

200

1000800

Time (min)

0200

400

11 hour perfusion; bile acids
n=5 refs taurocholate protocol

50250102030405060

Time after dosing (min)Figure 2. Effect of infusion of a (un)conjugated bile acid pool during 12 hours of NMP of porcine liver (n=3). (A) Perfusate cholesterol concentration intaurocholate (normal conditions) vs. Infusion of bile acids), (B) AST and (C ) ALT perfusate concentrations during perfusion (D) ICG clearance from theperfusate at 2 and 11 hours of perfusion compared to reference perfusions (taurocholate protocol).

2) Two ex vivo models were developed using porcine organs tostudy ADME processes of rosuvastatin and digoxin

4) Hepatic, biliary and renal clearance0.60.3050100150

Figure 3. Intestinal absorption ofrosuvastatin and digoxin. Papp values
in jejunum, ileum and colon of (A)Digoxin and (B) rosuvastatin. Data
represents mean ±SD (n=3).

0.60.3

JejunColonum0.0

Rosuvastatin
25201510500

Time after dosing (min)50100150

Time after dosing (min)D600200050100150

Time after dosing (min)Rosuvastatin (ng)40008000

BileUrine

digoxin (ng)Rosuvastatin perfusate (ng/mL)

5C

Bile Urine

Ileu

1000.9

BDigoxin
15Digoxin perfusate
concentration (ng/mL)

Perfusate

m

0.0

1.2m

Papp value (*10E-6 cm/s)

0.9

JejunA

Rosuvastatin

1.2um

Papp value (*10E-6 cm/s)

Digoxin

Ileu3) Intestinal transport• Porcine jejunum, ileum & colon tissue• Assessment of apical to basolateral transport• Cannulation hepatic artery & portal vein (liver) renal artery (kidney)• Slow bolus dosing via portal vein
• Perfusate, bile and urine samples taken over time

800

Time (min)Basolateral sampleb) Liver & kidney perfusion
Combined liver kidney perfusion was performed to study biliaryend renal clearance

6002 hour perfusion; bile acids

750

Human (n=4) and porcine (n=5) livers
underwent NMP for 360 min using taurocholate.
To study effect of the addition of (un)conjugated bile acids onliver viability and function, unconjugated bile acids (40% gCDCA,40% gCA, 10% CDCA, 10% CA) were infused for 12 hours of NMP(n=2)a) Intestinal regional transport
Intestinal transport of rosuvastatin and digoxin was determinedusing the InTESTine system

10030080000ICG clearance

ALTPerfusate ICG (%)

12h perfusion

ALT concentration (U/L)

1) During NMP of the liver the enterohepatic circulation ismissing, and standard protocols only
apply taurocholate1,2

2) Infusion of (un)conjugated bile acids during NMP
AST concentration (U/L)

METHODS

Figure 1. Changes in gene expression after hours of NMP in human (n=4) and porcine (n=5) livers. Genes related to (A) Cholesterol metabolism (B) Bile
acid synthesis and (C) Uptake and efflux transporters. Fold changes was calculated as expression relative to t=0h. Significance was measured by paired ttest, *p<0.05, **p<0.01, ***p<0.001.

Cholesterol delta (mg/dL)
t=60 vs t=end1) To optimize organ functioning during NMP by improving
physiological resemblance of the liver through the addition
bile acids2) To demonstrate prediction of ADME data of model drugs(rosuvastatin, digoxin) based on NMP of liver and kidneycombined with ex vivo intestinal absorption studies and PBPK
modeling.

Bile
Urine

6000
4000
2000
0050100

Figure 4. Hepatic andrenal clearance of digoxin
and rosuvastatin (A)Perfusate clearance ofdigoxin and (B)rosuvastatin. Biliary andurinary fractions weremeasured for (C) digoxinand (D) rosuvastatin. Data
represents mean ±SD(n=2).

150

Time after dosing (min)5) PBPK modellingc) PBPK modelling
A generic PBPK model was developed and combined with theinput of drug and system specific properties
Ex vivo models input:• InTESTine: regional
Papp data• Liver & kidney
perfusion derived data

REFERENCES

1. Eshmuminov, D et al., (2018). Perfusion settings
and additives in liver normothermic machine
perfusion with red blood cells as oxygen carrier.
A systematic review of human and porcine
perfusion protocols. Transplant
International, 31(9), 956-969.
2. Lascaris, B et al., (2022). Long-term
normothermic machine preservation of humanlivers: what is needed to succeed?. American
Journal of Physiology-Gastrointestinal and Liver
Physiology, 322(2), G183-G200.

Figure 5. Simulated
plasma profile of (A)digoxin and (B)rosuvastatin compared
to clinical data.

Table 1. The simulated vs. clinical pharmacokinetic parameters forsingle PO dose of 0.5 mg digoxin or 10 mg rosuvastatin• Ex vivo data was incorporated into a genericPBPK model and simulations resulted inrelatively accurate predictions of the plasma
concentration (Area Under the Curve (AUC)),
plasma peak concentration (Cmax), hepaticclearance and bioavailability when comparedto human clinical studies

CONCLUSIONS• 1) Portal infusion of (un)conjugated bile acids resulted in improved liver functions and cholesterol metabolism• 2) The combination of ex vivo gut, liver and kidney models with a generic PBPK model is a unique and powerful combination to predict
ADME profile of (new) drugs, with the possibility to calculate the fraction that undergoes enterohepatic circulation.
Future research is aimed at studying drug-drug interactions and the effects of disease processes.