PET TRACERS TO STUDY CLINICALLY
RELEVANT HEPATIC TRANSPORTERS
Andrea Testa,‡ Matteo Zanda, ‡ Charles S. Elmore, † Pradeep Sharma*††
‡
Kosterlitz Centre for Therapeutics, School of Medical Sciences, University of Aberdeen,
Foresterhill, Aberdeen, AB25 2ZD, UK
†
AstraZeneca R&D, Pepparedsleden 1, Mölndal, 431 83, Sweden
††
AstraZeneca R&D, Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, UK
KEYWORDS: Transporters, Hepatic, Positron Emission Tomography (PET), Imaging, DrugDrug Interactions (DDI)
*Corresponding author
DMPK, Drug Safety & Metabolism
AstraZeneca R&D
Unit 310, Darwin, Building, Cambridge Science Park
Milton Road, Cambridge, CB4 0WG
United Kingdom
E-mail: [email protected]
Phone: +44-(0)1625 234281
1
ABSTRACT
Transporter proteins expressed on the cell membranes of hepatocytes are directly involved in
the hepatic clearance, mediating the transport of drugs and metabolites through the hepatocyte,
from the blood stream into the bile. Reduction of hepatic transporter activity (due to chemical
inhibition, genetic polymorphism, or low expression) can increase systemic or liver exposure
to potentially toxic compounds, causing adverse effects. Many clinically used drugs have been
associated with inhibition of hepatic transporters in vitro, suggesting the potential involvement
of liver transporters in drug-drug interactions (DDIs).
Recently, radiolabeled hepatic transporter substrates have been successfully employed in
Positron Emission Tomography (PET) imaging to demonstrate inhibition of clinically relevant
hepatic transporters. The present article briefly describes the clinical relevance of hepatic
transporters followed by a review of the application of PET imaging for the determination of
pharmacokinetic parameters useful to describe the transporter activity and the design,
accessibility, preclinical and clinical applications of available radiotracers. Finally, based on
the analysis of the strengths and limitations of the available tracers, some criteria for the
development of novel PET probes for hepatic transporters and new potential applications are
suggested.
1. INTRODUCTION
Transporters are membrane-bound proteins that facilitate the vectorial movement of
endogenous or exogenous substrates across biological membranes.1-3
Transporters expressed in the liver have been shown to play critical roles in
pharmacokinetics,4 pharmocodynamics,5 drug-drug interactions (DDIs),6 targeted or site
specific deliveries,7 toxicological effects8 and therapeutic efficacy of drugs.9,10 Their impact
on environmental toxicology is another burgeoning area of research.11
2
The study of drug transporters involves the determination of binding affinities, transporter
efficiency, inhibition of transport, up/down regulation of transporter proteins, expression
and/or distribution patterns of novel putative carrier proteins, molecular mechanistic studies
of solute-carrier interaction and structure activity relationships of the transporter.
Various in silico,12 in situ,13 in vitro,14 and in vivo15 model systems are available to
accomplish these wide varieties of tasks. Each of these methodologies has advantages and
disadvantages. Traditional human pharmacokinetic mass balance studies, based on the
determination of the drug concentration in biological fluids (blood, plasma, serum, urine), do
not provide precise information about the actual drug concentration in tissues such as the
liver and kidneys. Employing a labeled drug, molecular imaging can be used for the noninvasive quantification of drug concentrations in clearance organs, allowing the
determination of pharmacokinetic parameters related to transporter activity.
Positron emission tomography (PET), because of its exceptional sensitivity and good spatial
resolution, is a powerful functional imaging technique that has been successfully used to
study molecular mechanisms behind numerous biological processes..
The current article briefly reviews the application of PET imaging in the determination of
pharmacokinetic parameters useful to describe the hepatic transporter activity and the design,
accessibility, preclinical and clinical applications of the available radiotracers. Finally, based
on the analysis of the strengths and limitations of the available tracers, some criteria for the
development of novel PET probes for hepatic transporters and new potential applications are
suggested.
3
2. APPLICATION OF PET TO STUDY HEPATIC TRANSPORTERS
The application of PET technology to study hepatic transporters could have certain distinct
advantages when compared to conventional in vitro cell assays and in vivo preclinical or
clinical PK studies. These are listed below:

PET imaging can be performed on small animals, large animals and humans

No physical collimators are required for coincidence counting with PET and it has a
higher sensitivity than other optical/nuclear imaging techniques

Since a PET tracer is typically used in microdose, it is unlikely to have
pharmacological or toxic effects

PET is a quantitative molecular imaging technique which can be used for kinetic
modeling applications. Accurate determination of the anatomical location of the probe
activity helps to identify areas of prime significance if the transporter is expressed at
multiple organs

PET has very low tissue attenuation (which can be corrected) of signal, hence no
depth limitation

The good spatial and temporal resolution of PET allows various physiological
phenomena (metabolism, transport and cell kinetics) to be imaged in a non-invasive
manner in living organisms at multiple time points.
Well known substrates of hepatic transporters have been labeled and employed to study
clinically relevant transporters. The biodistribution of these tracers has been studied with or
without the co-administration of selective inhibitors of transporters. The following are the
major reported applications of PET imaging in the area of hepatic transport.
2.1 Determination of the uptake hepatic clearance and the apparent liver to blood AUC
Mathematical models called “integration plots” have been designed, in order to calculate the
hepatic clearance and the canalicular efflux clearance by measuring the concentration of the
4
tracer in the plasma and in tissues of interest such as liver and intestine. The tissue
compartments are generally assumed to be well stirred, with a rapid equilibrium between the
capillary bed and the interstitial fluid.16
While the tracer concentration in the blood can be monitored by measuring the radioactivity
in blood samples collected during the imaging experiment, the concentrations of the tracer in
the liver and intestine can be monitored via non-invasive PET imaging.
Liver and intestine are selected as volumetric regions of interest (ROIs), and the radioactivity
concentration, as fractions of the total injected radioactivity, can be measured over time. As
demonstrated by Takashima et al.,17 the sum of the radioactivity in gallbladder and intestine
can be considered equal to the radioactivity in the bile secreted to these tissues. This was
proved by comparing the radioactivity measured in the intestine via PET imaging with the
radioactivity found in bile samples obtained from bile duct cannulas. To consider the
contribution of the partial volume effect from the bile excreted to the intestine, the ROIs for
this tissue were defined to include a portion of the nearby regions.
As proposed by Takashima and Watanabe,18 the hepatic uptake rates of the radiotracer can be
calculated from the measure of the radioactivity in the liver and in the blood. Within short
periods of time after PET tracer injection (generally 1-3 minutes), the metabolism of the
tracer can be considered negligible and the efflux can be assumed much smaller than the
influx. The hepatic uptake clearance CLuptake is determined from the linear equation (1)
𝑋𝐻(𝑡)
𝐶𝑏𝑙𝑜𝑜𝑑(𝑡)
= 𝐶𝐿𝑢𝑝𝑡𝑎𝑘𝑒
𝐴𝑈𝐶𝑏𝑙𝑜𝑜𝑑 (0−𝑡)
𝐶𝑏𝑙𝑜𝑜𝑑 (𝑡)
+ 𝑉0
(1)
where X H(t) is the amount of radioactivity found in the liver at the time t, Cblood(t) is the
amount of the radioactivity in the blood at the time t, AUCblood (0-t) is the area under the blood
concentration-time curve from 0 to t, and V0 is the initial distribution volume in the liver at
time 0. Plotting [X H (t)/Cblood (t)] versus [AUCblood (0-t)/ Cblood(t) ], V0 is the y intercept and
CLuptake the slope of the straight line.
5
It is also possible to calculate the apparent liver-to-blood AUC ratio, Kp, liver (2), which
represents the liver exposure of the radiotracer and is a reflection of the hepatic extraction
ratio:
𝐾𝑝,𝑙𝑖𝑣𝑒𝑟 =
𝐴𝑈𝐶𝐻(0−𝑡)
𝐴𝑈𝐶𝑏𝑙𝑜𝑜𝑑 (0−𝑡)
(2)
Where AUCH(0-t) is the area under the hepatic concentration-time curve from time 0 to t. If Kp
> 1, the tracer is likely to be a substrate of uptake transporters. The significant differences can
be found for Kp and CLuptake values between control groups and hepatic transporter-inhibitor
treated groups.
2.2 Determination of the biliary clearance
The biliary clearance of the radiotracer can be determined in a non-invasive way as proposed
by Takashima et al.18 by measuring the amount of radiotracer in the liver and the amount of
the radiotracer excreted into the intestine through the bile. In fact, the accumulation of the
radioactivity in the bile can be described by the linear equation (3):
𝑋𝑏𝑖𝑙𝑒 (𝑡) = 𝐶𝐿𝑖𝑛𝑡,
𝑏𝑖𝑙𝑒
∙ 𝐴𝑈𝐶𝐻 (0−𝑡) + 𝑉𝑒
(3)
Where Xbile(t) is the amount of radioactivity found in the bile (intestine), CLint, bile is the
canalicular efflux clearance of the radiotracer, and Ve the y intercept of the straight line.
If the radiotracer undergoes hepatic metabolism during the imaging experiment, the number
of metabolites and their relative amounts should be determined in order to calculate the
contribution of each metabolite to the total amount of radioactivity measured by the PET scan
in the intestine. In this case, the intrinsic biliary clearance of each metabolite can be
determined from the equation (3). If the metabolites pattern is unknown, the same equation
can be used to calculate the canalicular clearance of the total radioactivity associated to the
PET tracer and its metabolites.
6
As a reflection of the uptake from the systemic circulation and the efflux from the canalicular
membrane, the biliary secretion clearance, CLbile, can be calculated using the following
equation (4):
𝐶𝐿𝑏𝑖𝑙𝑒 =
𝑋𝑏𝑖𝑙𝑒 (0−𝑡)
𝐴𝑈𝐶𝑏𝑙𝑜𝑜𝑑 (0−𝑡)
(4)
where X bile (0-t) is the amount of radioactivity secreted in the bile between time 0 and t.
3. PET TRACERS TO STUDY HEPATIC TRANSPORTERS
The ideal PET tracer to study hepatic transporters should exhibit the following
characteristics:

Actively and selectively transported by specific uptake and/or efflux transporters

Show low/no passive diffusion through cellular membranes

Metabolically stable

Safe to administer in vivo

Efficiently and easily labeled with a PET isotope having half-life long enough to
allow the imaging of the clearance process

Easily formulated for intravenous dosing
In reality it might be difficult to find a PET tracer with all the aforementioned attributes. If
the tracer is employed to study the uptake process in hepatocytes, the absence of hepatic
metabolism is not strictly required, but the transport should not be blood-flow limited in order
to observe the effect of transporter inhibitors on the tracer uptake. On the other hand, if
observing the biliary efflux process is the objective of the study, high metabolic stability of
the tracer is desirable, and the biological half-life of the radiolabeled tracer should be
comparable with the physical half-life time of the labeled radioisotope.
Clinically used drugs that undergo hepatic excretion are common starting points for the
design of a PET tracer for the study of hepatic transporters, because their pharmacokinetics
7
and toxicology have already been extensively studied throughout the drug development
process. Also, drug metabolites17,19 and endogenous compound derivatives20,21 have been
successfully labeled with 11C and employed as PET tracers. Table 1 lists the PET tracers that
have been used to study hepatic transporters.
8
Table 1. PET tracers that had been used to study hepatic transporters.
Used preclinically
or clinically
(species)
References
[11C]-Dehydropravastatin OATPs, MRP2
Preclinical (rat)
22,23
[11C]-Rosuvastatin
OATPs, NTCP, BCRP
Preclinical (rat)
24
[11C]-TIC-Me
OATP1B1, OATP1B3,
MRP2
Preclinical (rat)
and clinical
18,25
[11C]-Glyburide
OATPs
Preclinical(baboon,
mouse)
26
[11C]-Telmisartan
OATP1B3
Preclinical (rat)
and clinical
27-29
[11C]-Metformin
OCT1, MATE1
Preclinical (mouse,
rat, pig)
30,31
[11C]-Rhodamine-123
OCT1, P-gp
Preclinical (rat,
mouse)
32
[11C]SC-62807
OATPs, BCRP
Preclinical (mouse)
17,19
[11C]-Cholylsarcosine
Bile acid transporters
Preclinical (pig)
20
[11C]-N-acetyl-cysteinyl-
OATPs, MRP2
Preclinical (rat,
monkey)
21
PET tracer
Hepatic transporters
studied
leukotriene E4
The following sections describe the synthesis and in vitro characterization of the reported
tracers employed to study hepatic transporters, along with a discussion of the in vivo imaging
results. In most cases, in vitro studies have been performed in cells expressing human
transporters, while in vivo experiments were conducted with rodents or other animal models
(pigs, baboons). This practice is generally acceptable, considering the high functional
homology between human and rodent transporters. However, during the interpretation of the
results, one should take into account possible specific differences in the substrate recognition
of human and murine transporters.
9
3.1 [11C]-Dehydropravastatin as a PET tracer to study OATPs and MRP2 in rats
Dehydropravastatin 1 (DPV) is an analogue of the lipoprotein-lowering drug pravastatin 2
which was developed to evaluate the functions of OATP1B1 and MRP2 in vivo.22
In comparison to pravastatin, dehydropravastatin lacks the stereogenic centre at the C2’
carbon and the presence of the double bond between the C2’ and C3’ allowed the 11C labeling
via cross coupling chemistry (Figure 1). The slight difference of molecular structure between
DPV 1 and pravastatin 2 is postulated to have a negligible effect on the potential of DPV to
be an OATPs and MRP2 substrate.
Figure 1. Chemical structures of dehydropravastatin 1 and pravastatin 2
The synthesis of [11C]-1 was accomplished via a palladium mediated Suzuki coupling of the
organoboron precursor 3 and [11C]-CH3I, followed by deprotection of the hydroxyl groups
concomitant with lactone ring opening and formation of the sodium salt (scheme 1).23
Scheme 1. Reagents and conditions: (i) [11C]-CH3I, Pd2(dba)3, P(o-tolyl)3, K2CO3, THF, 65
°C, 5 min; (ii) NBu4F, 60 °C, 2 min; (iii) 0.1 M NaOH, r.t., 1 min.
10
The uptake of DPV was examined in vitro using freshly prepared rat hepatocytes and rodent
MRP2-expressing membrane vesicles. The kinetic transport parameters for DPV were found
similar to those obtained for pravastatin 2. In the presence of rifampicin (an OATPs inhibitor)
the uptake of DPV by hepatocytes was completely inhibited, supporting the hypothesis that
DPV, like pravastatin, is a substrate of uptake transporters like OATPs. DPV and pravastatin
were also found to inhibit the MRP2 transport of estradiol 17β glucuronide (E217βG), a probe
substrate of MRP2.
The metabolism of [11C]-1 was studied in rats and human cryopreserved hepatocytes. While
no metabolism was observed within 90 minutes of incubation with human hepatocytes, at
least two major metabolites were found in the blood, liver and bile of rats after the
intravenous administration of [11C]-1.
Time-plasma concentration profile, hepatic uptake, and canalicular efflux clearance of [11C]1 were studied in control, rifampicin-treated, and MRP2-deficient rats (Eiasi
hyperbilirubemic mutant rat). Within 5 minutes of the intravenous injection of [11C]-1, the
radioactivity had cleared from the blood in all the animal models. After the same period of
time, the elimination in rifampicin-treated and MRP2-deficient rats was significantly reduced.
[11C]-1 extensively accumulated in the liver, in which the uptake was blood-flow limited,
with a maximum peak at about 2 minutes after administration. In rifampicin-treated and
MRP2-deficient rats the AUCs of radioactivity in the liver were 1.8 and 3- fold larger than in
the control rats. The maximum amount of radioactivity in the intestine was found to be 2.5
and 4- fold less in rifampicin-treated and MRP2-deficient rats than in control rats,
respectively. The hepatic uptake clearance and the canalicular efflux clearance were also
calculated for [11C]-1 using the integration plot method. As expected, the hepatic uptake
clearance in MRP2-deficient rats was similar to the control rats, while the canalicular efflux
clearance was about 9-fold smaller. In rifampicin-treated rats, the hepatic uptake clearance of
11
[11C]-1 was only slightly smaller than the value found in control rats. This was probably due
to the blood-flow limited hepatic uptake of [11C]-1. The canalicular efflux clearance was 2
fold smaller in rifampicin-treated rats than in control rats, demonstrating the inhibitory effect
of this molecule on the activity of MRP2.22
3.2 [11C]-Rosuvastatin as a PET tracer to study OATPs and MRP2 in rats
Rosuvastatin 5 is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, clinically
used for the treatment of dyslipidemia. Because rosuvastatin is a known substrate of the
transporters OATPs, NTCP, MRP2, and BCRP,33-36 the [11C]-isotopomer [11C]-5 was
developed to study the hepatobiliary transport of the drug by means of quantitative PET
imaging.24
[11C]-5 was prepared by N-methylation of the desmethyl precursor 4 with [11C]-CH3I in the
presence of tetrabutylphosphonium hydroxide (scheme 2). After HPLC purification, [11C]-5
was obtained in 54% decay-corrected yield in about 35 minutes.24
Scheme 2. Reagents and conditions: (i) [11C]-CH3I, PBu4OH, MeCN/DMSO, 105-120 °C, 5
min.
In a preclinical study, [11C]-5 was administered in rats intravenously (i.v. bolus dose of
1.41µg/kg, 1 mCi) over 30 s with and without rifampicin (40 mg/kg i.v. bolus plus 1.85
mg/min/kg infusion).24 It was observed that [11C]-5 selectively accumulated in the liver and
kidneys. In the rat, rosuvastatin is transported into the liver by uptake transporters OATPs
12
(1a1, 1a4, 1a5, and 1b2), but not by NTCP.34 In the liver, it is biotransformed by metabolising
enzymes and/or excreted by MRP2 and BCRP.36,37 In the kidneys, rosuvastatin is actively
taken up by the renal transporter OAT3.38 The co-administration of rifampicin increased
systemic AUC by 3-fold, while it decreased accumulation (AUC 0-15min) of [11C]-5 in the liver
and kidneys by 54% and 73%, respectively. The study showed that [11C]-5 is a sensitive PET
tracer for studying the inhibition of hepatic transporters by rifamipicin in rats, and the tracer
may be a useful PET probe for human hepatic transporters. By co-administration of inhibitor
drugs with [11C]-5 by i.v. and oral route separately, it might be possible to understand the
impact of gut transporters in rosuvastatin DDIs. The use of pitavastatin as a PET probe might
be a useful strategy as this molecule has recently been shown to be a more specific and
sensitive clinical probe for OATP1B1 inhibition when co-administered with rifampicin in
humans.39
3.3 [11C]-TIC-Me as a PET tracer to study OATP1B1-OATP1B3 and MRP2 in rats and
humans
(15R)-TIC-Me, (15R)-16-m-tolyl-17,18,19,20-tetranoisocarbacyclin methyl ester, 6 was
originally developed as a radioligand for the imaging of prostacyclin receptor PGI2 in the
thalamus.40
15-(R)-TIC-Me 6 is a prodrug of the pharmacologically active acid 15-(R)-TIC 7 (Figure 2)
that is subjected to hepatic clearance.18 Due to the structure similarity between (15R)-TIC and
prostaglandin E2, which is a well-known OATP substrate, it was postulated that [11C]-6
could be a good tracer to study hepatobiliary transport in vivo.
13
Figure 2. Chemical structures of (15R)-TIC 7 and its prodrug (15R)-TIC-Me 6.
The 11C labeling was achieved by using a Stille coupling of stannane 8 and [11C]-CH3I
(scheme 3).41
Scheme 3. Reagents and conditions: (i) [11C]-CH3I, Pd2(dba)3/P(o-tolyl)3 (1:4), CuCl, K2CO3
DMF, 65 °C, 5 min.
In vitro assays were performed in order to study the uptake of [3H]-(15R)-TIC in OATP1B1,
OATP1B3, OATP2B1, NTCP-expressing HEK239 cells, control cells, and human
hepatocytes.18 The OATP1B1 and OATP1B3 contributions to the hepatic uptake of [3H](15R)-TIC were calculated by fractional functional contribution study using reference
substrates for OATP1B1 and OATP1B3.
14
In the hepatic uptake of (15R)-TIC, the contributions of OATP1B1 and OATP1B3 were
69.3% and 30.7%, respectively. No difference between the transport of [3H]-(15R)-TIC in
OATP2B1 and control cells were found, suggesting that the molecule is not a substrate of that
transporter. Although the authors did not find a sodium dependence in the transport of [3H](15R)-TIC in human hepatocytes, indicating that NTCP is not involved in the transport, it
was observed to be transported in NTCP expressing cells. The contribution of NTCP in the
transport of [3H]-(15R)-TIC was considered negligible if compared to the contribution of
OATPs.
The metabolism of the PET tracer [11C]-6 was thoroughly investigated in normal and MRP2deficient rats. In neither of them the methyl ester prodrug could be detected in the blood 1
minute after the administration. The carboxylic acid [11C]-7 was detectable for up to 5
minutes after injection. At least three metabolites were detected in the blood, liver and bile of
normal and MRP2-deficient rats. The three major metabolites were identified as a beta
oxidation (de-ethylation) product of [11C]-7 (M1), the acylglucoronide of [11C]-7 (M2), and
the acylglucoronide of M1 (M3). Most of the radioactivity found in the liver of MRP2deficient and control rats was derived from the metabolite M2, while in the bile, the most
abundant metabolite was M3 in both animal models.
The hepatobiliary transport of [11C]-7 and its metabolites were extensively investigated in
normal and MRP2-deficient rats. After 2 minutes from the injection of [11C]-(15R)-TIC-Me,
the radioactivity was mainly located in the liver and kidneys. After 60 minutes, most of the
radioactivity was localised in the intestine of normal rats while no signal was detected in the
intestine of MRP2-deficient rats. The AUCliver0-90 was 2.7 fold higher in MRP2 deficient rats
than in control rats.
The hepatic uptake clearance of [11C]-6 was calculated by means of the integration plot
method, using the equation 1. The value found for control rats (45 mL/min/Kg) was
15
comparable with the rat’s hepatic blood flow rate (55 mL/min/Kg), suggesting a very
effective hepatic uptake of [11C]-(15R)-TIC. In MRP2-deficient rats, the hepatic uptake
clearance of [11C]-6 was slightly reduced, probably due to the lower OATP expression 42 and
to the high bilirubin glucuronide concentration, which may inhibit the uptake of OATP
ligands.43
The intrinsic biliary clearance values of the two metabolites found in the bile, M2 and M3,
were calculated using the equation (2).
For the major metabolite found in the bile, M3, the canalicular efflux clearance CLint, bile, M3
was decreased to 14% in MRP2 deficient rats, while, for the metabolite M2, the reduction of
the CL int, bile, M2 was not statistically significant, probably because of the involvement of other
efflux transporters in the excretion of M2 from the hepatocyte. This was supported by in vitro
assays employing MRP2 and BCRP expressing vesicles: M2 and M3 were good substrates of
MRP2, while only M2 was significantly transported by BCRP.
In humans, a similar metabolic profile pattern was found,25 with the conversion of [11C]-6 to
the free carboxylic acid [11C]-7 in less than 2 minutes and the formation of M1, M2, and M3.
A new metabolite was also found, but its structure was not clarified in the study. The
hepatobiliary transport of [11C]-6 was then studied in healthy human volunteers with and
without rifampicin treatment.
After 17 minutes from the intravenous administration of [11C]-6, the maximum levels of
radioactivity were found in the liver in both control and rifampicin treated subjects. In
rifampicin treated subjects the AUC blood (0-30) was 1.5-fold higher than in control subjects.
Consequently, the amount of radioactivity in the bile excreted into the intestine of rifampicintreated subjects was reduced to 50% of the control.
The hepatic uptake clearance of [11C]-7 was calculated as described before by employing the
equation (3). The value found in control subjects ranged from 46% to 79% of the blood flow
16
rate. At the dose employed in the study, rifampicin was able to reduce the hepatic uptake
clearance by 45%, while no effects were observed on the renal uptake clearance. This was in
agreement with the Ki values of rifampicin found for OATP1B1 and OATP1B3 (0.62 μM and
0.39 μM, respectively) and the concentration of unbound rifampicin present in the systemic
circulation (1.3 μM) after 1 hour of administration of a 600 mg dose. The systemic
concentration of rifampicin was higher than the Ki values, therefore it could produce a
detectable inhibition of transporters activity.
Even though the effects of rifampicin on the efflux clearance were variable among the
subjects, a significant reduction of the canalicular efflux of the radioactivity (by 62%) was
observed in rifampicin treated subjects, probably due to the inhibition of the MRP2
transporter.
[11C]-6 was successfully employed as a PET tracer to quantitatively study the activity of the
rat transporter MRP2 and, due to its history of clinical use, it was safely employed in humans
to assess the effect of rifampicin on the hepatobiliary functionality. In this valuable work, the
authors proved the use of PET imaging to analyse the kinetics of the hepatobiliary transport
in humans and demonstrated that, at the clinical dose, rifampicin is able to inhibit the hepatic
uptake via OATP1B1 and OATP1B3.
3.4 [11C]-Glyburide as a PET tracer to study OATP1B1 in baboons
Glyburide 10, a sulphonylurea receptor 1 inhibitor, is a widely prescribed anti-diabetic agent,
used in the treatment of type 2 diabetes. It is a substrate of several ABC transporters (ATP
Binding Cassette proteins) (BCRP, MRP1 and MRP3)44,45 but is also a substrate of
OATP1B1, OATP2B1 and OATP1A2.46,47
[11C]-Glyburide [11C]-10 was obtained via methylation of the desmethyl precursor 9 with
[11C]-CH3OTf at 110 °C for 2 minutes (scheme 4).26
17
Scheme 4. Reagents and conditions: (i) [11C]-CH3OTf, 3N NaOH, Acetone, 110 °C, 2 min.
In vitro studies were performed to measure the uptake of [3H]-glyburide in P-gp and BCRPexpressing MDCKII cells, with or without the presence of FTC (a BCRP inhibitor),48,49
PSC833 (a P-gp inhibitor)50,51 and GF 120918 (a dual P-gp/BCRP inhibitor).52 The uptake of
[3H]-glyburide in P-gp expressing MDCKII cells was significantly increased by the presence
of PSC833 and GF120918, while the effect of the FTC was negligible. Similarly, the uptake
of [3H]-glyburide in BCRP-expressing MDCKII cells was significantly increased by the
presence of FTC and GF120918, whilst the specific P-gp inhibitor PSC833 did not affect the
uptake.
PET imaging and metabolism experiments were performed in anesthetised baboons with or
without the co-administration of pantoprazole (CYP3A4 inhibitor), rifampicin (OATPs
inhibitor), and cyclosporine A (OATPs/ABC transporters and CYP3A4 inhibitor). No
metabolites of [11C]-10 were found in blood when rifampicin or cyclosporine A were coadministered, suggesting that the transport of glyburide from the blood stream to the liver is
the first key step in glyburide metabolism.
[11C]-Glyburide accumulated in the liver of control baboons, showing the hepatobiliary
extraction of the labeled drug. The rifampicin and cyclosporine A treatments significantly
reduced the liver-to-blood AUC ratio, Kp, liver, by 14 and 18- fold, respectively. As a
consequence of the decreased hepatic uptake, the [11C]-glyburide exposure to the renal
cortex, myocardium, pancreas and lungs, increased. Regarding the biliary excretion, no
significant results were obtained due to great variability in the PET imaging experiments.
18
This might be due to the relatively short time period investigated (60 minutes) compared to
the glyburide half-life (4.7 hours in humans). The results confirmed that OATP transporters
control the glyburide biodistribution and have an important impact on its metabolism. As no
difference was found between the rifampicin and cyclosporine A treatments, the impact of Pgp inhibition in the biodistribution of glyburide was believed to be minor, at least when
OATP was already inhibited.
3.5 [11C]-Telmisartan as a PET tracer to study OATP1B3 in rats and humans
Telmisartan 12 is an angiotensin receptor antagonist used in the management of hypertension.
It is a lipophilic compound mainly transported into the liver by the OATP1B3 transporter.53
[11C]-Telmisartan [11C]-12 was selected as a PET tracer to study the activity of OATP1B3
and was prepared by the coupling of desmethyl precursor 11 with [11C]-CH3I, followed by
methyl ester hydrolysis (scheme 5). An automated synthesis was also developed for the
clinical production of [11C]-12.28
Scheme 5. Reagents and conditions: (i) 1M KOH, [11C]-CH3I DMSO, 120 °C, 5 min, (ii) 1M
NaOH, 100 °C, 3 min.
The metabolism of [11C]-12 was investigated in normal rats. No metabolites were found in
the blood after 20 minutes of the administration, while in the liver, telmisartan was
transformed into the glucuronide metabolite by UDP-glucuronyltransferases.54 The biliary
excretion of telmisartan-glucuronide (the sole metabolite of the drug) was observed.
19
The hepatobiliary transport of [11C]-12 was at first studied in normal and rifampicin-treated
rats, then in healthy human volunteers.27,29,55 In control rats, a maximum of the radioactivity
was observed at 14 minutes post-injection and, from that point, the radioactivity was
observed to accumulate gradually in the intestine. In rifampicin treated rats, the radioactivity
observed in the liver decreased in a dose dependent manner while the radioactivity in the
intestine was unaffected. Compared to control rats, the AUC0-90 blood was 2.4 and 3.9 fold
higher in rats treated with a constant infusion of rifampicin at 0.5 and 1.5 μmol/min/Kg
respectively.
The hepatic uptake clearance CLuptake calculated by the integration plot method, was found to
be very close to the hepatic blood flow in control rats, but it was possible to observe a
significant reduction of CLuptake to 65% in rats treated with rifampicin at 1.5 μmol/min/Kg.
No significant difference was found in the biliary efflux clearance (CLint, bile) between control
and rifampicin treated rats.
The effect of the administration of 1, 4, and 10 mg/kg of unlabeled telmisartan on the
biodistribution of [11C]-12 was also investigated. The elimination of the radioactivity from
the systemic circulation was reduced in a dose dependent manner. The hepatic uptake
clearance of [11C]-12 was significantly reduced to 52% at the dose of 10 mg/kg of unlabeled
telmisartan. On the other hand, at the dose of 4 and 10 mg/kg, unlabeled telmisartan increased
the biliary efflux of total radioactivity probably due to up-regulation of efflux transporters.
A clinical study was performed on healthy human volunteers in order to determine the
biodistribution and radiation dosimetry of [11C]-12 in humans.27 After few minutes from
[11C]-12 injection, the kidneys, intestine and other organs were weakly visible. Subsequent
imaging confirmed the hepatobiliary clearance of the tracer by accumulating at first in the
liver and gradually eliminating into the gallbladder and the intestine. The liver was the organ
in which the highest level of radioactivity was observed, with a peak of 55.7±3.3% of the
20
injected dose at 0.80 h. The effective dose for [11C]-12 was found comparable to the doses of
other 11C tracers published in literature, and no adverse effects, changes in physical
conditions or abnormal findings were observed after the termination of the scan. The tracer
was thus found safe and promising in order to study OATP1B3 in humans.
3.6 [11C]-Metformin as a PET tracer to study OCT1 and MATE1 in mice, rats and pigs
Metformin 15 is a biguanide drug widely employed in the treatment of type II diabetes.56 Its
site of action is the hepatocyte, in which it inhibits two fundamental enzymes involved in the
gluconeogenesis: glucose-6-phosphatase and phosphoenolpyruvate carboxykinase.57
Metformin is transported into the hepatocyte via the OCT1 transporter58 and is then excreted
unchanged into the bile via the efflux transporters MATE1 and MATE2K.59
While [14C]metformin has been generally employed as a probe substrate for in vitro testing of
OCT1 activity, the 11C labelled form of metformin for PET imaging was first published in the
literature in 2013.30 The synthesis was initiated by reacting [11C]-CH3I with methylamine to
give [11C]-dimethylamine [11C]-13 which was subsequently treated with cyanogen bromide to
afford [11C]-cyanamide [11C]-14. Reaction of [11C]-14 with guanidine hydrochloride at high
temperature in the presence of sodium hydroxide gave [11C]-15 in an overall 17 %
radiochemical yield (scheme 6).
Scheme 6. Reagents and conditions: (i) 2M MeNH2 in THF, Dimethylacetamide, 45 °C, 5
min; (ii) BrCN, IPr2NEt, Dimethylacetamide, 45 °C, 5 min; (iii) guanidine hydrochloride,
NaOH, H2O, 175 °C, 5 min
21
The biodistribution of [11C]-15 was studied in pyrimethamine (an inhibitor of MATE1 and
OCT1)-treated and control mice by injecting the compounds into the tail vein.30 In both the
animal groups, the accumulation of radioactivity was observed in the kidneys and the urinary
bladder. The radioactivity in the liver of control mice rapidly decreased after 2 minutes. In
pyrimethamine-treated mice, an enhanced accumulation of the radioactivity was observed in
the liver, which reached a peak between 6 and 7 minutes after the administration. This effect
was due to the effective inhibition of the extrusion transporter MATE1 by the presence of
pyrimethamine, reducing the canalicular efflux of the radioactivity. Negligible effects on
OCT1 inhibition were observed in the imaging experiment because at the concentrations
employed in the study (5 mg/kg, corresponding to unbound concentration 0.3 µM),
pyrimethamine has no effects on OCT1 (Ki =3.6 µM), while it is able to inhibit MATE1 (Ki=
0.14 nM). Although the work did not describe the impact on human renal transporters (OCT2
and MATE1/2K) it is proposed that it may also be possible to use [11C]-15 to study the
activities of OCT2 and MATEs in the kidney.
In a different study, the biodistribution of [11C]-15 was also investigated in rats and pigs.31 In
both the animal models the activity accumulated in liver and kidneys. Administration of cold
metformin delayed the hepatic and renal clearance.31
3.7 [11C]-Rhodamine-123 as a PET tracer to study OCT1 and P-gp in rats
Rhodamine 123 17 is a weakly basic fluorone dye, known to accumulate in the mitochondria.
It has also been employed in oncology research in order to measure the P-gp expression in
certain drug resistant cancer cells60-63 and at the blood-brain-barrier.64,65 In vitro experiments
22
using OCT1-expressing HEK293 cells demonstrated that rhodamine 123 is a high-affinity
substrate for both the organic cation transporters.66
[11C]-Rhodamine-123 [11C]-17 was synthesised by esterifying rhodamine 110 16 with [11C]CH3I (scheme 7).32
Scheme 7. Reagents and conditions: (i) [11C]-CH3I, NBu4OH, DMF.
The tracer was stable for at least 2.5 hours in the plasma of rats, but some metabolites, whose
structures were not resolved, were found during in vivo experiments. The biodistribution of
[11C]-17 was investigated with PET in different groups of mice; wild type, P-gp knock out,
OCT1/2 knockout and cimetidine pre-treated P-gp knock-out mice. The higher accumulation
of [11C]-17 in the kidneys of wild type mice compared to that of OCT1/2 knock out mice
confirmed that [11C]-17 is a good in vivo substrate for OCT1/2 transporters as observed in
vitro.66 There was no difference in time-activity profile of [11C]-17 in the kidneys of wild
type and P-gp knock-out mice, indicating that there is no involvement of P-gp in the kidneys
distribution of [11C]-17. Pre-treatment of wild type rats with DCPQ (((2R)-anti-5-{3-[49,10,11-dichloromethanobibenzosuber-5-yl)piperazin-1-yl]-2-hyroxypropyl}quinoline
trihydrochloride) a P-gp inhibitor, resulted in greater peak radioactivity in the liver,
confirming the role of P-gp in the biliary excretion of [11C]-17 in rats. Pre-treatment of rats
with cimetidine increased the systemic concentrations of [11C]-17, which may be due to
inhibition uptake in the kidneys by renal transporters, as described previously, and/or due to
23
inhibition of metabolic pathways by cimetidine in the liver.67,68 Since no data is available to
compare accumulation of [11C]-17 between cimetidine treated and untreated rat liver, it is
difficult to conclude what is the exact role of OCT1 in the liver uptake of [11C]-17.
3.8 [11C]SC-62807 as a PET tracer to study OATP1B1, OATP1B3 and BCRP in mice
SC-62807 20 is the principal metabolite of the selective cyclooxygenase inhibitor celecoxib.
It is produced in the liver by two-step oxidation of celecoxib and is then excreted in the bile
by the BCRP.69
[11C]SC-62807 was prepared via a Suzuki reaction of [11C]-CH3I and boronate ester 18 to
give [11C]-celecoxib [11C]-19 which was oxidized to [11C]-SC-62807 [11C]-20 by means of
basic potassium permanganate under microwave irradiation (scheme 8).17
Scheme 8. Reagents and conditions: (i) (a) [11C]-CH3I, Pd2(dba)3, P(o-tolyl)3, DMF, 65 °C, 4
min. (ii) KMnO4, 0.2 M NaOH, 140 °C (microwave), 5 min.
In vitro studies were performed to evaluate the uptake of [11C]-20 in OATP1B1 and
OATP1B3-expressing HEK 239 cells, human and rodents BCRP vesicles, and human
hepatocytes.
[11C]-20 proved to be a good substrate for rodent and human BCRP, OATP1B1, and
OATP1B3 transporters. The relative contributions of OATP1B1 and OATP1B3 transporters
in the human hepatocyte uptake were found to be similar. Metabolism and imaging studies
were performed in wild-type and BCRP knock-out mice.19 No metabolites of [11C]-20 were
24
found in the blood, bile, liver and urine of normal and BCRP deficient mice. After 2 minutes
from the injection of [11C]-20, the radioactivity was mainly located in the liver and kidneys of
both animal groups. By 30 minutes, most of the radioactivity was localized in the intestine
and in the urinary bladder of normal mice whereas the AUCblood (0-30) was 2.2- fold higher in
BCRP-deficient mice, indicating that the transportation of the radioactivity in BCRPdeficient mice had been slowed.
Biliary secretion clearance (equation 4) and canalicular clearance (equation 3) were
significantly lower in BCRP deficient mice than in control mice (10 and 4- fold,
respectively). Similar results were found for the renal secretion clearance and the kidneybrush-border efflux clearance, whose values were found 25 and 100-fold lower in BCRPdeficient mice, proving that BCRP is essential in the biliary and renal excretion of anionic
drug metabolites such as SC-62807.
The authors also conducted a study to assess the capability of [11C]-20 to study BCRP
function at the Blood Brain Barrier (BBB). Unfortunately, low uptake of the tracer into the
brain was observed in both BCRP-deficient and control mice probably because of the low
membrane permeability of [11C]-20.
3.9 [11C]-Cholylsarcosine as a PET tracer to study bile acid transporters in pigs
Cholylsarcosine 23 is an analogue of the bile acid conjugated cholyl-glycine. It is a
metabolically stable derivative of cholic acid and the amino acid sarcosine (N-methylglycine)
and is subjected to enterohepatic circulation in humans.20,70
[11C]-Cholylsarcosine [11C]-23 was prepared by N-methylation of the methylester of glycine
with [11C]-CH3I and subsequent coupling with cholic acid. The [11C]-cholylsarcosine
25
methylester so obtained was purified by HPLC and then hydrolysed with aqueous NaOH to
give [11C]-cholylsarcosine [11C]-23(scheme 9).20
Scheme 9. Reagents and conditions: (i) (a) [11C]-CH3I, 1,2,2,6,6-pentamethylpiperidine,
DMSO, 50 °C, 5 min. (ii) diethyl cyanophosphonate, cholic acid, DMSO, 50 °C, 5 min. (iii)
0.25 M NaOH, room temperature. 2 min.
Because of the high similarity between cholylsarcosine and cholylglycine, it has been
postulated that cholylsarcosine is a substrate of NTCP and BSEP transporters. However no in
vitro transport studies have been performed as yet and the contribution of other transporters
such as OATPs and BCRP in the hepatobiliary transport of this bile acid analogue cannot be
excluded.
Imaging experiments were performed in pigs to investigate the hepatocellular transport and
biodistribution of [11C]-23.20 The tracer rapidly accumulated in the liver (peak within 3
minutes post-injection) and was then excreted into the intra-hepatic and common bile duct
and eventually accumulated in the gallbladder or intestines. As a consequence of
enterohepatic circulation, the radioactivity in the liver and bile ducts increased again 75
minutes after the injection. The imaging experiment was repeated in one of the animals after
administration of cholyltaurine, an endogenous bile acid conjugate (286 mg/ Kg), to
investigate the inhibition of the hepatobiliary transport of [11C]-23. Inhibition of both hepatic
uptake and biliary excretion was observed, with a substantial reduction of the liver AUC and
choledochus AUC, and a much slower transportation rate of the radioactivity into the
intestine. The binding of [11C]-23 to plasma proteins was found to be very similar to that of
26
endogenous cholylderivatives (60-80%). It was also demonstrated that cholyltaurine
displaced [11C]-23 from plasma proteins, meaning that [11C]-23 binds to the same proteins as
endogenous bile acids. The hepatobiliary transport of a bile acid analogue and the effects of
alterations of bile acid transporters were analysed in a non-invasive way, showing how the
imaging of hepatic transporters could be employed in the characterization of normal and
pathological liver functionality.
3.10 [11C]-N-acetyl-cysteinyl-leukotriene E4 for the study of OATP1B1 and MRP1/2 in
rats and monkeys
N-acetyl-cysteinyl-leukotriene E4 (LTE4NAc) 25 is the major metabolite of endogenous
cysteinyl leukotrienes in rodents.71 Leukotrienes, produced from arachidoic acid in the
lipoxygenase pathway, are important mediators of inflammatory response72 and are generally
substrates of OATP1B1 and MRP1/273. In order to assess the contribution of different organs
to the clearance of leukotrienes, a 11C-labeled version of LTE4Nac, [11C]-25, was prepared
and its biodistribution was studied in normal rats, transporter deficient mutant rats (with a
hereditary defect in the leucotriene elimination),74 and monkeys.21
[11C]-Acetyl chloride [11C]-24 was obtained by reaction of [11C]-carbon dioxide with
methylmagnesium chloride and subsequent treatment with phthaloyl chloride. The acetylation
of LTE4 was then performed in the presence of 2,6-dimetylpyridine to afford [11C]-LTE4NAc
[11C]-25 after HPLC purification (scheme 10).75
27
Scheme 10. Reagents and conditions: (i) CH3MgCl, THF, -78 °C. (ii) phthaloyl chloride,
THF, 80 °C, 5 min. (iii) leukotriene E4, 2,6-dimethylpyridine, THF, 45 °C, 10 min.
To the best of our knowledge, no in vitro transport studies have been performed to investigate
the transport kinetics of LTE4NAc 25 on specific transporters. Metabolism of 25 was studied
in normal rats, transporter deficient mutant rats (impaired bilirubin secretion),76 and
cholestatic rats, employing the tritium labelled [3H]-LTE4NAc. Major metabolites were polar,
shorter-chain, oxidation products. The biological half-life of [3H]-LTE4NAc was about 40
seconds in normal rats and about 85% of the administrated radioactivity was excreted into the
bile within 1 hour.21 In the same animal model, the amount of radioactivity found in urine
was about 4%. In transporter deficient mutant rats 6% of the administrated radioactivity was
excreted in the bile and 21% in the urine within 1 hour, whereas in cholestatic rats a delayed
renal elimination of the radioactivity was observed.21
PET imaging of [11C]-25 showed that the radioactivity accumulated in the liver (with a
maximum activity concentration in the liver at 4 minutes for control rats, and 9-10 minutes
for transporter mutant and cholestatic rats) and then in the intestine of normal rats or in the
urinary bladder of mutant and cholestatic rats. The mean liver transit time was found to be 17
minutes for normal rats and 54 minutes for transporter mutant rats.
In monkeys, as in normal rats, the hepatobiliary elimination exceeded the renal clearance of
[11C]-25, with a maximum liver activity at 12 minutes post-injection and mean liver transit
time of 34 minutes.21
This pioneering PET study showed how, in the case of impaired hepatic functionality (due to
hereditary transporter deficiencies or bile duct obstruction), the extraction of leukotriene
metabolites can be switched from hepatobiliary to renal clearance.
28
4. DESIGN OF PET TRACERS FOR HEPATIC TRANSPORTERS
Although PET has been extensively used in various areas of drug discovery and development
in the past decades, its use to study the involvement of hepatic transporters in drug
pharmacokinetics is a relatively new but burgeoning area of research. In principle,
incorporation of a PET isotope into the structure of most drug molecules to study hepatic
transport should be feasible; however there are significant technical challenges to overcome,
mainly due to the short half-lives of PET tracers. While for decades, the very short half-life
times of 11C and 18F, have limited the chemical space of PET tracers to few entities, now the
evolution of novel synthetic methodologies virtually allows the labeling of any existing drug.
The development of 11C carbonylation methodology, for example, allows the introduction of
the radioisotope in metabolically stable carbonyl, carboxyl functions and heterocyclic
compounds.77 Fluorine-18 radiochemistry has expanded considerably as well,78 leading to the
development of new strategies that, in principle, could allow the late stage radiofluorination
of almost all the fluorinated clinically used drugs.
Ex-novo design of completely new hepatic PET probes would involve a multidisciplinary
approach consisting of specific syntheses based on structure activity relationships,
radiolabeling, in vitro and in vivo evaluation and kinetic modeling of the radiolabeled
compound before it is applied to preclinical or clinical research (Figure 3). The key medicinal
chemistry principles for designing hepatoselective PET tracers can be generated based on in
silico and computational modelling of substrates and /or inhibitors of major uptake
(OATP1B1/1B3, NTCP, OCT1) and efflux (P-gp, BCRP, MATE1, MRP2) drug transporters.
In order to design hepatoselective PET probes for uptake transporter(s), the following
attributes are desirable:
29

Structural features - Several studies have shown that incorporation of an acidic moiety
(anionic) is a useful strategy to enable recognition of compounds by uptake
transporters like OATP1B1 /1B3.79,80 Inclusion of a cationic tertiary amine could
favour uptake by the OCT1 transporter. For NTCP inhibitors, pharmacophores
consisting of hydrophobic regions and one hydrogen bond acceptor have been
proposed.81,82

Molecular weight (M.W) and relative polar surface area (RPSA) – M.W. of  400 Da
and RPSA  20% have been observed with known OATP substrates.80

Low passive permeability - Poor permeability of compounds prevents diffusion of the
probe from hepatocytes back into sinusoidal blood capillaries and is helpful in terms
of intracellular accumulation of the PET probe. Generally, a membrane permeability
value of 10-6 cm/s or lower, as determined from in vitro transcellular assays, is
classified as low permeability of a compound.83

Low lipophilicity (logD at pH7.4  2) - Most of the known good substrates of
OATP1B1/1B3 have low logD values.84 Although, low logD is the preferred property
to impart hepatoselectivity to compounds, very low logD may have adverse ADME
issues. Tu et al79 had analysed hepatoselective properties of a large set of diverse
compounds from varied chemical spaces against their physiochemical and
pharmacokinetic properties. The authors observed that the optimal lipophilicity for
orally administered hepatoselective drugs lies between logD of 0.5 to 2.0.

Optimum solubility - High solubility of a PET probes is desirable in order to enable a
higher fractions of the dose to be absorbed after oral administration. However, if the
PET tracer is developed for intravenous administration, then this attribute may not be
critical.
30
There is a considerable overlap of substrates between uptake and efflux transporters,80 and
because of this they share a similar sets of important molecular descriptors for design of
optimal PET tracers. However, there might be subtle differences in the desirable values of
these molecular descriptors required for good substrates of efflux transporters, when
compared to uptake transporters. For example, the structural features, logD and the
membrane permeability value requirements for substrates of efflux transporters require them
to be more lipophilic85 than substrates of uptake transporters. The molecular descriptors
proposed for P-gp substrates are a logP value of  2.92,  18-atom long molecular axis, high
energy of the highest occupied orbital and at least one tertiary basic nitrogen atom.85
Similarly, quantitative structure activity relationships (QSAR) had been reported and
extensively reviewed for BCRP,86 MATE1/2K,87 MRP2,88 and BSEP89 transporters.
Although a detailed analysis of QSAR for each hepatic transporter lies outwith the scope of
this review, the key findings concerning these aspects can be helpful stepping stones for the
design of completely novel hepatic PET tracers.
As evident from sections 4.0, most hepatic PET probes reported in the literature so far are
well established drugs, drug metabolites or their synthetic derivatives. This obviates the need
to establish safety margins for them if they are used at micro-dose or sub-therapeutic dose
levels in PET studies. Their transporter kinetics are well characterized in in vitro and/or in
vivo models and readily available from the literature. In principle, instead of sticking to the
prototypical probe drugs already reported in literature, it might be possible to study any drug
of interest as a “victim” by labeling it with a PET isotope. Labeling a drug and coadministering it with the investigational “perpetrator” drug would not only enable the study
pairs of drugs, but also to study in the patient population of interest, instead of normal healthy
volunteers. Also, because only trace doses of a PET probe are needed, drugs with narrow
therapeutic index (for example, paclitaxel) could be used as PET tracers.
31
5. VALIDATION, DESIGN AND INTERPRETATION OF PET STUDIES
The validation of PET tracers as tools for studying hepatic transport may be done prior to
their recommendation as in vivo probes for routine drug-drug interaction studies (Figure 3).
This validation should involve full transporter kinetics of the PET tracers in in vitro models
and determination of apparent Michaelis-Menten kinetics (Km), maximal transport velocity
(Vmax), active clearance (Clactive) and passive clearance (Cldiff ). If multiple transporters are
involved then kinetics for each transporter should be evaluated. These constants, along with
physicochemical properties (logP, pKa, B/P ratio, PPB), would be valuable if full
physiologically based pharmacokinetic (PBPK) modeling of data generated from the PET
study is desired. An in vitro transporter inhibition assays using a PET tracer as probe
substrate will help to gauge the sensitivity of the PET tracer for in vivo DDI studies. The
determination of metabolic clearance (Clin) in human liver microsomes or similar systems
and identification of generated metabolite(s) is necessary to aid the interpretation of in vivo
findings in a PET tracer studies. A preclinical PK study of the PET tracer will help to
establish its distribution and elimination pathways. A DDI study in preclinical species using
the PET tracer as the probe and a known potential inhibitor can provide results on the
efficacy of use of the PET tracer as suitable probe. In vitro transporter inhibition results can
be correlated to in vivo findings to validate the model. Although there are no direct orthologs
between human and rodent hepatic OATP isoforms, which despite these fundamental
differences are functionally comparable at a collective level.90 Thus, preliminary DDI studies
in preclinical species using PET probes still offer an important preparatory step before taking
the probe into clinical studies.
Most PET probes for hepatic transporters studied so far lack specificity in transport mechanism
and are also eliminated by renal transporters (Table 1). For example, [11C]-rosuvastatin is not
32
only eliminated by hepatic (OATPs, MRP2 and BCRP) but also by renal transporters (OAT3).
Since it is possible to get concentration versus time profiles of specific organ systems in a PET
study, ‘integration plots’ from these concentration profiles can provide data on changes in
concentrations/distribution at specific organs in the presence of a “perpetrator” candidate drug.
This will not only help to identify level of interactions at organ levels, but also provide a
mechanistic interpretations of the interaction. For example, after intravenous administration of
[11C] -Rhodamine-123 to wild-type rodents, PET and ex vivo measurements showed that
radioactivity uptake was very low in brain, but relatively high in some other organs such as
heart, and especially liver and kidneys. Inhibition of P-gp increased uptake in brain, heart,
kidney and liver, but only by up to twofold. Secretion of radioactivity from kidneys was
markedly reduced by OCT knockout or pretreatment with Cimetidine. [11C]-Rhodamine-123
was unsuitable as a PET probe for P-gp function and appears to be a strong substrate of OCT1/2
in kidneys. Cimetidine appears to be effective for blocking OCT1/2 in kidneys in vivo. This is
especially advantageous in light of the fact that most in vitro models cannot simultaneously
study the effect of all transporters involved in the PK of a “victim” drug and cannot provide
information on whole body PK when co-administered with a “perpetrator” drug. However, PET
cannot distinguish the individual contribution of specific transporters co-located in the same
organ. For example, [11C]-(15R)-TIC is actively transported into the liver and PET
demonstrated Cluptake,liver of 21 ml/min/Kg in humans and the tracer-related radioactivity
reached 37% of dose in the liver within 17 min of intravenous administration.25 However, it
could not be confirmed from the PET study how much of this uptake clearance is contributed
by OATP1B1 and/or OATP1B3. Therefore, relative activity factor (RAF) or fractional
functional contribution was determined in vitro using HEK293 expressing OATP1B1 or
OATP1B3 transporters along with human hepatocytes.25
33
The complete knowledge of elimination mechanisms and their relative contribution to the
overall elimination of the proposed hepatic PET probe can provide useful information about
the principal component involved in an observed DDI with the probe.91 For example, [11C]rosuvastatin absorption is affected by BCRP and the systemic concentrations are controlled by
active hepatic (OATP1B1, OATP1B3, NTCP) and renal uptake (OAT3). When administered
by oral or intravenous route, rosuvastatin can be used as probe for hepatic uptake transporters
(OATP1B1 and OATP1B3), intestinal efflux transporters (BCRP) and /or renal uptake
transporter (OAT3). The visualization of the PET probe and changes in local accumulation
pattern will help identifying the actual transporter(s) implicated in any observed DDI.
While PET studies are easy to perform in conscious humans, they are difficult to perform in
moving preclinical species. Often this requires to sedate animals with suitable anesthetics. This
might interfere with normal physiology of the body and introduce artifacts in PK observations
by PET in preclinical species.
Another major challenge in the interpretation of data from hepatic PET studies is the
biotransformation of probes by action of metabolizing enzymes to generate various fractions
of metabolites containing PET isotopes. This makes the determination of the mass balance of
the parent PET tracer relative to the metabolites difficult, unless additional metabolite
profiling studies are performed (which might also involve withdrawing blood samples at time
points to know the exact concentrations of radiometabolites if formed at appreciable levels).
The interest in the imaging of hepatic transporters, especially OATP1B1 and OATP1B3,92 is
not limited to the field of DMPK: recently OATPs have been recognized as potential
biomarkers for gastrointestinal,93 breast,94 prostate,95 pancreas96 and lung97 cancers.
Moreover, OATPs, for which expression is often altered in abnormal cells, play an important
role in hormone98,99 and drug distribution in cancer cells.97 A selective PET probe for OATP
expression would allow for a better understanding of cancer cell metabolism.100
34
Moreover, the use of PET tracers like [11C]-cholylsarcosine [11C]-21 transported by bile acid
transporters, NTCP and BSEP, could potentially provide valuable insight as diagnostic agents
for pathophysiological hepatic conditions and also for drug induced hepatotoxicities which
produce a perturbation of these transporters.
6. CONCLUSIONS
The in vivo PET imaging of hepatic transporters is still an emerging technology that allows
the quantification of critical kinetic parameters useful to describe the activity of such
transporters.
The effects of chemical inhibition and reduced activity of specific hepatic transporters on the
pharmacokinetic profile of reference drugs have been successfully studied, prompting the
development of standard imaging protocols that can be adopted in drug safety studies.
The mechanism of some DDIs has been clarified and unequivocally attributed to inhibition of
transporters.
A deeper understanding of the function of transporters in the liver and in tissues in which
they are abnormally expressed would be also useful for the diagnosis of liver diseases and
cancers.
35
Figure 3. Schematic representation of the development process of a PET tracer to study hepatic transporters.
Selection of PET tracer
Optimisation of PET tracer suitability
Application
In vitro studies
Ideal PET tracer for hepatic transporter
•High affinity specific substrate of transporter
•Do not metabolise
•Non-toxic
•Feasible radiochemical synthesis
•Physicochemical stability and amenable to intravenous
formulation
•Michaelis-Menten kinetics (low Km) in in vitro
transporter model
•In vitro inhibition of transport (low IC50) with
clinical perpetrator drug
• Negligible metabolism
• Plasma protein binding (PPB) and log P
determination to help build physiologically based
pharmacokinetic (PBPK) model
Preclinical or clinical application
• Determination of DDIs due to hepatic
transporter inhibition
•Determination of hepatotoxicity due to
inhibition of bile acid transporters
In vivo studies
DDI studies in rodents
DDI studies in humans
36
ACKNOWLEDGEMENT
The critical review of manuscript by Katherine Fenner, DMPK DSM, AstraZeneca Ltd. UK
is appreciated. The work for this review article is supported by grants from AstraZeneca Ltd.
UK and SINAPSE (Scottish Imaging Network), Scotland, towards PhD studentship of AT.
PS and CE are employees of AstraZeneca Ltd., UK.
ABBREVIATIONS
AUC, area under the curve; DDI, drug-drug interaction; DMF, dimethylformamide; DMSO,
dimethyl sulfoxide; DPV, dehydropravastatin; HPLC, high-performance liquid
chromatography MRI, magnetic resonance imaging; PET, positron emission tomography;
PK, pharmacokinetic; RAF, relative activity factor; ROI, region of interest; RPSA, relative
polar surface area; THF, tetrahydrofuran.
37
TABLE OF CONTENTS GRAPHICAL ABSTRACT
Blood Stream
Hepatocyte
[11C]-Metformin
OCT1
MATE1
[11C]-Metformin
BCRP
Bile
BSEP
MRP2
P-gP
[11C]-SC-62807
OATP1B1
[11C]-Cholylsarcosine
OATP1B3
[11C]-Tic-Me
[11C]-SC-62807
[11C]-TIC-Me
[11C]-Rhodamine
[11C]-Cholylsarcosine
NTCP
38
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