Discussion
Here we demonstrate for the first
time the use of NMP of explanted diseased human livers and the
applicability for pharmacokinetic research. We successfully perfused 7
cirrhotic livers and 4 non-cirrhotic livers for a period of 360 min,
maintaining liver viability and functionality, as indicated by stable
flow, bile production, albumin synthesis, bilirubin excretion, proper
histology pre- and post-perfusion and stable ALT and AST values.
Additionally, the model showed to be useful to study hepatic first pass,
clearance, biliary excretion and drug-drug interaction.
The use of NMP has shown to be of great benefit in the field of organ
transplantation (11, 17). Over the past years, NMP has become a widely
accepted method to assess viability of the donor liver prior to
transplantation (18, 19). Many criteria of hepatocellular and
cholangiocellular function have been described (e.g. lactate clearance,
perfusate AST, perfusate glucose levels and biliary pH, bicarbonate and
glucose levels) to establish liver viability based on perfusion results
and post-transplantation outcomes, which demonstrates the robustness of
the model in perfusion research (13-17). The explanted cirrhotic livers
perfused in this study met most of the criteria for hepatocellular
function, except for lactate clearance and portal flow whereas other
hemodynamic parameters did not show to be significantly affected. As
expected, portal flow was lower in cirrhotic livers compared to
non-cirrhotic livers as a result of portal hypertension (20).
Cholangiocellular function remained intact during 360 min of NMP as
shown by the presence of glucose resorption and bicarbonate excretion.
We previously showed the
application of NMP of porcine livers to predict drug-drug interaction on
plasma exposure of commonly prescribed statin drugs (21). The degree of
drug-drug interactions derived from the NMP model were in good agreement
with clinical data showing no effect of ischemia-reperfusion injury on
the pharmacokinetics of drugs. Literature is scarce regarding human
models that predict drug pharmacokinetics especially in cirrhotic
conditions. In the current study we demonstrate the application to study
hepatic first pass, clearance and biliary excretion in a perfused
explanted human liver model. This approach showed to give comparable
results with published in vivo human data. In patients with liver
cirrhosis, differences can arise in portal vein concentration after oral
dosing due to differences in intestinal absorption and/or portal
hypertension compared to individuals without liver cirrhosis. However,
in this study we administered the same bolus to the portal vein to study
differences in hepatic first pass and clearance between the cirrhotic
and non-cirrhotic livers. The non-cirrhotic livers showed to rapidly
take up rosuvastatin and digoxin from the perfusate with a hepatic
extraction ratio of 0.74 and 0.40 respectively which are close toin vivo reported measures of 0.63 and 0.3 respectively (22, 23).
In this study, hepatic first-pass and clearance of rosuvastatin and
digoxin showed to be the most affected by cirrhosis. Rane et al. (24)
reported that the clearance of hepatically cleared drugs with a high
extraction ratio are related to blood flow and thus a major decrease in
portal flow as in cirrhosis can dramatically affect the first passage of
across the liver (25, 26). In addition, in vivo studies
demonstrated a high biliary excretion of rosuvastatin of approximately
76.8% as measured by fecal excretion (27). The ex vivonon-cirrhotic livers showed a biliary excretion of 37% in 120 min,
extrapolation of the data resulted in 77% total excretion of
rosuvastatin which is in line with in vivo data. Interestingly,
digoxin showed a relative high biliary clearance in cirrhotic (51%) and
non-cirrhotic ( 17%) livers during 120 min of perfusion. In vivostudies have shown that digoxin is extensively renally eliminated (75%)
(28). However, multiple studies demonstrated that digoxin is highly
involved in the enterohepatic circulation, thereby decreasing thein vivo fecal excretion of digoxin (29, 30). The two other
compounds used in this study, furosemide and metformin, which are mainly
renally cleared, showed a low hepatic extraction ratio and minor biliary
excretion (≤3%) in both cirrhotic and non-cirrhotic which is in line
with human in vivo data which showed that biliary eliminated was
limited (31, 32). Interestingly, the percentage of biliary clearance was
higher, for all compounds, in the cirrhotic perfused livers. This might
be due to an elevated bile flow which has been observed in patients
cirrhosis and which is confirmed in our model, resulting in a more
efficiency biliary clearance (33).
The effect of drug-drug interactions in cirrhotic and non-cirrhotic
livers was subsequently determined by using a cocktail of inhibitors.
The non-cirrhotic livers showed an increased AUCR for a drug-drug
interaction with rosuvastatin (3.52) where values between 2.48 – 5.38
for rosuvastatin with rifampicin as inhibitor have been observed
(34-36). Rosuvastatin is mainly inhibited at the hepatic level, since it
is an OATP substrate, and therefore the perfused liver model is showing
to properly predict the degree of drug-drug interaction for
rosuvastatin. Digoxin, showed a high increase in AUCR upon dosing with
inhibitors, which is potentially the result of inhibiting uptake via
OATP2 (rifampicin as inhibitor) as well as biliary secretion via Pgp
(quinidine as inhibitor). However, this is difficult to compare toin vivo observations since s major part of the drug-drug
interaction takes place at the intestinal level, when orally absorbed,
thereby affecting the portal vein concentration. Still, the observations
from this study showed that we could mimic a drug-drug interaction with
digoxin in this perfusion model leading to an increased Cmax and AUCR.
Explanted livers obtained during orthotopic liver transplantation are
currently only used for pathological assessment and subsequently
discarded. While many preclinical and laboratory animal models try to
mimic liver diseases as best as possible, many models fail due to a lack
of translation to the human situation. Despite the advantages of
utilizing explanted livers, such as specific disease characteristics,
maintained hepatic architecture and presence of systemic inflammation
and collagen content, this NMP explanted liver model is not widely
adopted (or has not been reported) (4). Considering the frequency of
orthotopic liver transplantation, and the ease of combining this with
the relative simple procedure to prepare the liver for perfusion, this
model can be widely applied in a variety of research settings (37). For
the field of drug development, use of perfused explanted livers might
not only contribute to the reduction in the use of laboratory animals,
but more importantly enable better predictions on drug pharmacokineticsin vivo . It will increase our knowledge on hepatic blood flow,
hepatic first pass effect and biliary clearance of drugs under normal
and cirrhotic conditions. Besides changes in blood flow and collagen
content, changes in pharmacokinetics of drugs in patients with liver
disease might also be the result of alterations in expression levels of
transporter proteins and/or metabolic enzymes. In fact, multiple studies
have analyzed liver biopsies from patients with liver disease showing
alterations in expression of specific proteins relevant for
pharmacokinetics (38-41). For instance, Drozdzik et al., showed an
increase in Pgp and multidrug resistance protein 4 (MRP4) and decreases
in NTCP, OCT1 and OATP1B1 in patients with severe liver disease (39) .
Although these studies already provided some hints towards altered
pharmacokinetics and metabolism of drugs in patients with liver
diseases, ex vivo perfusion of diseased livers offers a unique
opportunity to directly study the effect of altered expression levels of
transporter proteins and metabolizing enzymes. In this study we used
known drug substrates for different important hepatic uptake and efflux
transporters. Gaining insight into pharmacokinetic profiles of
OATP1B1/1B2, Pgp, BCRP and OCT-1 model compounds is a first step towards
studying transporter functions in diseased liver. This information can
be easily extrapolated to other drugs which are substrates for one of
the transporters assessed in this study. Additionally, for many drugs,
dosing advice is currently incomplete for patients with cirrhosis
because of lacking evidence or showing major interindividual
differences. Studying drug pharmacokinetics using explanted human livers
may substantiate dosing advice for this group of patients (42).
In addition to using cirrhotic liver perfusion to study drug
pharmacokinetics, this model provides new possibilities to study
specific liver functions under different pathological circumstances for
multiple applications, e.g. disease specific biomarkers. Currently,
there are several examples of using machine perfusion platforms to study
organ reconditioning, pharmacological interventions to prevent
ischemia-reperfusion injury, defatting steatotic livers or studying the
effect of oncolytics (43-47). Recent studies have adapted machine
perfusion to demonstrate the possibility to prolong organ perfusion
duration (48-50), with perfusion of human and porcine livers for up to 7
days (18). This might lead to increased knowledge regarding disease
pathology and enable the ex vivo treatment of discarded or
explanted livers with medication or stem cells (51). Such future
interventions would certainly increase the availability of suitable
donor organs.
In conclusion, we demonstrated for the first time stable NMP of diseased
human livers explanted during liver transplantation and discarded donor
livers to study hepatic first pass, clearance ,biliary excretion and
drug-drug interactions. This approach provided comparable results to
published in vivo human data supporting its applicability as a
robust ex vivo drug handling model.
Acknowledgements: We thank Elwin Verheij, René Braakman and
Pieter Spigt for their help with the LCMS method development of the drug
cocktail.