List of abbreviations
ALD Alcoholic liver disease
ALT Alanine aminotransferase
AST Aspartate aminotransferase
AUC Area under the curve
AUCR Area under the curve ratio
CIT Cold ischemia time
Cmax Maximum concentration
HA Hepatic artery
HTK Histidine-tryptophan-ketoglutarate
HBV Hepatitis B virus
HCC Hepatocellular carcinoma
MELD Model for end stage liver disease
NAFLD Non-alcoholic fatty liver disease
NMP Normothermic machine perfusion
OLTx Orthotopic liver transplantation
PBC Primary biliary cholangitis
PBPK Physiologically based pharmacokinetic modelling
PV Portal vein
WIT Warm ischemia time
Introduction
Accurate prediction of drug disposition in patients with and without
hepatic diseases remains difficult, as appropriate models are lacking.
The liver plays an important role in drug handling and impairment or
alteration of its function may greatly affect multiple processes. Upon
first liver pass, after oral administration, drug bioavailability as
well as drug clearance may be altered thereby affecting the drug’s
efficacy. Studies in liver
cirrhosis have shown that increased bioavailability as well as reduced
clearance lead to a higher prevalence of adverse drug reactions and
drug-drug interactions which can result in safety issues and ultimately
an increased risk for hospital admission (1, 2). Therefore, drug dosing
should be tailored according to the varying degree of liver dysfunction
among patients with liver diseases. However, with the currently
available preclinical and clinical models, it remains difficult to
quantify the required tailoring of the dose related to the degree and
type of liver dysfunction (3).
Established in vitro and animal models are often used to study
the pathology and pharmacological characteristics of drugs of varying
diseases. However, translation of these findings to clinical practice
remains challenging due to, among others, species differences in
transporter expression and the difficulty to mimic dynamic liver
processes (4, 5). Novel 3D models like liver-on-a-chip and bile
duct-on-a-chip models have gained significant interest as a predictive
platform to study liver processes due to the incorporation of
haemodynamics (6, 7). Although these organ-on-a-chip models hold much
promise, they are still in their infancy owing to the difficulty of
mimicking (patho)physiological processes in the liver such as portal and
arterial blood flow and biliary excretion (7).
Normothermic machine perfusion
(NMP) systems using human ex vivo whole organs overcome this
problem since hepatic architecture is combined with (near) physiological
hemodynamics. Thereby, use of human explanted liver whole organ enables
to study hepatobiliary processes as well as liver disease specific
pharmacokinetics (8-10).
In this feasibility study we explored the applicability of using
explanted human diseased livers for NMP research. Besides standard liver
function assessment, we explored liver functionality by studying drug
pharmacokinetics; hepatic first-pass, clearance and biliary excretion of
four model compounds (rosuvastatin, digoxin, furosemide and metformin).
These compounds are known substrates for different important hepatic
uptake and efflux transporters. We also studied drug-drug interactions
of the model drugs with a cocktail of perpetrator drugs.
Materials and Methods
Human livers
Patients undergoing liver transplantation were included in this study.
After providing informed consent, the patients approved the usage of the
explanted liver for experimental study. The use of explanted liver
tissue was approved by the medical ethical committee of the Leiden
University Medical Center (B19.040). Patients with polycystic liver
disease, with a transjugular intrahepatic portosystemic shunt (TIPS), or
waitlisted for recurrent- orthotopic liver transplantation were excluded
from participation. Eleven human livers were included in the study. The
underlying disease processes of these livers were primary biliary
cholangitis (PBC, n=1), non-alcoholic fatty liver disease (NAFLD, n=2),
alcoholic liver disease (ALD, n=3), hepatocellular carcinoma in the
context of Hepatitis B viral disease (HBV+HCC, n=2). In addition, three
discarded non-cirrhotic livers which were declined for transplantation
were included in this study. The reasons for decline were; steatosis
(n=2) and a occlusion of right hepatic artery (n=1). Immediately
following explantation of the recipient diseased liver, a portal and
arterial flush with cold Histidine-tryptophan-ketoglutarate (HTK)
(Carnamedica, Warsaw, Poland) preservation solution was performed. The
period between explantation (i.e. clamping and transection of the portal
and hepatic veins as final step of the hepatectomy) and cold flush of
the explanted liver (ex vivo ), is described as the warm ischemia
time (WIT). After a clean effluent flush, the liver was transported in
cold preservation solution to the Organ Preservation and Regeneration
room in the OR complex. Here, under sterile conditions, a back table
reconstruction of the right and left hepatic artery and portal vein was
performed using surplus donor blood vessels, in order to facilitate
cannulation (portal vein- 25Fr cannula, hepatic artery – 12Fr cannula)
and connection to the machine perfusion device
(LiverAssistTM device, XVIVO, Groningen, the
Netherlands). Thereafter, the bile duct was cannulated in order to
collect bile fractions during perfusion. Additional flush of the hepatic
artery and portal vein was performed on the back table.
Normothermic machine perfusion
All human livers were perfused using the LiverAssistTMdevice. The machine consists of two centrifugal pumps which provide a
pulsatile flow to the hepatic artery and a continuous flow to the portal
vein (11). The system reservoir was filled with 2L perfusion fluid
containing 1:1 ratio of 4x packs of human red blood cells and 4x packs
of fresh frozen plasma (Sanquin, Amsterdam, the Netherlands). Insulin,
sodium taurocholate, heparin and epoprostenol were provided as
continuous infusion at a rate of 10U/h, 1041U/h, 10 mL/h (2% w/v) and 8
µg/h, respectively, in order to maintain liver functioning and to
facilitate bile flow. Additionally, nutrients (aminoplasmal 10E (B Braun
Melsungen AG, Melsungen, Germany) and cernevit (Baxter BV, Utrecht, the
Netherlands) were continuously provided (23mL/hr) to keep the liver
metabolically active (Supplemental table 1). Gas delivery to the
LiverAssistTM consisted of 95% oxygen and 5% carbon
dioxide at 1.5 L/min and the temperature was set at 37°C. The
non-cirrhotic livers were perfused with a portal pressure of 11 mmHg and
the cirrhotic livers required perfusion at 14 mmHg to generate a
sufficient portal flow. Mean arterial pressure was set at 50 mmHg. Upon
perfusion, additional boluses of sodium bicarbonate and glucose were
given when necessary to maintain physiological pH
(~7.35-7.45) and glucose (>5mmol/L) levels.
After 360 minutes of perfusion, the livers were submerged in
formaldehyde and transported the pathology department and were examined
according to the institution’s clinical guidelines dependent on the
patient’s underlying pathophysiology.
Drug administration during perfusion
Drug clearance in perfusate and bile of the drug cocktail (rosuvastatin,
metformin, furosemide and digoxin) were determined in the absence and
presence of drug inhibitors (quinidine, rifampicin, cimetidine and
probenecid). The selection of the drug cocktail applied in this study
was based since the cocktail is representative for different important
hepatic and renal uptake and efflux transporters (12). The dosage
applied to the system was based on the typical oral dosage and was
corrected for the fraction absorbed in the intestine to the portal vein,
fraction of metabolism and circulating volume which is shown in
supplemental table 2. After 120 min. of perfusion, a slow bolus for 10
min of the drug cocktail was administered via the portal vein at 1mL/min
to mimic oral absorption through the gut. Subsequently, perfusate and
bile samples were taken for the following 120 min. Arterial samples were
taken at t=120, 122, 124, 126, 128, 130, 135, 140, 150, 160, 170, 180,
210, and 240 min. Additional portal samples were taken during the
administration of the drug at t=126 and t=130 min to determine the
hepatic extraction. Bile samples were collected in 10 minute fractions
from 120 min onwards. After 240 min., first a slow bolus 10 min
(1mL/min) of inhibitor mix (quinidine, cimetidine, rifampicin and
probenecid) was administered to the liver and after 5 min (at t=245
min.), again, a subsequent slow bolus of the drug cocktail was
administered via the portal vein. The same sampling schedule for
arterial samples and bile samples was followed. Perfusate and bile
samples were immediately stored at ≤-70°C until further processing.
Liver function assessment
Hepatic artery and portal vein flow were recorded from the
LiverAssistTM machine. Arterial and venous perfusate
samples and bile samples (collected under mineral oil to prevent bile
exposure to ambient air (13)) were taken hourly to monitor liver
viability (pH, glucose, lactate etc.) using a RapidPoint 500 blood gas
analyzer (Siemens, Germany). The total bilirubin, alanine
aminotransferase (ALT) and aspartate aminotransferase (AST)
concentration in the perfusate samples was measured by reflectance
photometry (Reflotron-Plus system, Roche diagnostics, Almere, the
Netherlands). Perfusate and bile parameters were compared to defined
criteria used in clinical transplantation studies; perfusate ALT
<6000 and lactate <2.5 mmol/L after 120 min of
perfusion, biliary pH >7.5, Δ glucose perfusate – bile
<10 mmol/L, Δ bicarbonate bile – perfusate >0
mmol/L (14-16).
Histological analysis
Pre-perfusion (n=2) and post-perfusion (n=2) biopsies were taken for
each liver, fixed in 10% formalin and subsequently embedded in
paraffin. Slices of 4µm were cut and stained with hematoxylin & eosin
(H&E) or Sirius red for examination using light microscopy.
Bioanalysis
The concentration of the drug cocktail was quantified using LC-MS/MS
(Waters, Etten-Leur, the Netherlands). Perfusate and bile sample (10 µL)
were deproteinized with 100 µL acetonitrile (ACN) with the addition of
10 of µL the isotopically labelled internal standards (1µg/mL).
Thereafter samples were vortexed, centrifuged and supernatant was
transferred to 96 well plate and dried under nitrogen. Thereafter,
samples were dissolved in 100 µL 10% ACN + 0,1% formic acid and
injected in to LC-MS/MS for quantification. Details of the LC-MS/MS
conditions used are shown in supplemental table 3.
Chemicals
Rosuvastatin, digoxin, furosemide, quinidine were obtained from
Sigma-Aldrich (Zwijndrecht, the Netherlands). Metformin and rifampicin
and cimetidine were obtained from Bioconnect (Huissen, the Netherlands).
Heparin, sodium taurocholate (Sigma-Aldrich, Zwijndrecht, the
Netherlands), insulin (Novo Nordisk, Alphen aan den Rijn, the
Netherlands) and epoprostenol (Flolan; GlaxoSmithKline Inc, Mississauga,
ON, Canada) were obtained as indicated.
Data analysis and statistics
Data obtained during the perfusion studies was analyzed using Prism
version 8 (GraphPad, California, USA). Values for the area under the
concentration time curve 0 -120 min (AUC0-tau) were
calculated using the linear trapezoidal method. The area under the
concentration time curve ratio (AUCR) was determined by dividing the
AUC125-245 min (with inhibitors) by the
AUC0-120 min (without inhibitors). The hepatic
extraction ratio was calculated during the 10 min dosing period as
following: concentration entering the liver (portal vein) -
concentration leaving the liver / concentration entering the liver.
Significance of differences between the cirrhotic and non-cirrhotic
livers was tested using the Mann-Whitney U test. Data is presented as
median and inter-quartile range (IQR) for non-parametric distributed
data. P-value below 0.05 was considered significant.