pet studies in drug development: methodological considerations
TRANSCRIPT
TECHNOLOGIES
DRUG DISCOVERY
TODAY
PET studies in drug development:Methodological considerationsVincent J. Cunningham1,*, Christine A. Parker2, Eugenii A. Rabiner2,
Antony D. Gee2, Roger N. Gunn1
1Translational Medicine and Genetics, GlaxoSmithKline, 891-995 Greenford Road, Greenford, Middlesex, UK UB6 7HE2Translational Medicine and Genetics, GlaxoSmithKline, ACCI, Addenbrookes Hospital, Hills Road, Cambridge, UK CB2 2GG
Drug Discovery Today: Technologies Vol. 2, No. 4 2005
Editors-in-Chief
Kelvin Lam – Pfizer, Inc., USA
Henk Timmerman – Vrije Universiteit, The Netherlands
Imaging technologies
*Corresponding author: V.J. Cunningham ([email protected])
1740-6749/$ � 2005 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2005.11.003
Section Editor:Adriaan A. Lammertsma – Department of Nuclear Medicineand PET Research, Vrije Universiteit Amsterdam,The Netherlands
Positron emission tomography (PET), as an in vivo
pharmacological imaging tool in experimental medi-
cine, is playing an increasing role in drug development.
There are two areas where PET is particularly useful in
this respect, namely biodistribution and drug occu-
pancy studies. Radiotracer design, the properties of
the molecular targets which can be studied and the
quantitative estimation of pharmacological endpoints
will be discussed in relation to these applications, with
particular reference to studies in brain.
Introduction
In addition to the study of underlyingmechanisms of disease,
there are currently two areas where positron emission tomo-
graphy (PET) is particularly useful and can have a direct
impact on the drug development process, namely biodistri-
bution and drug occupancy studies.
For biodistribution studies, the drug itself is radiolabelled.
PET can then provide quantitative information on its deliv-
ery and turnover in specific tissues following intravenous
administration to the subject under study. The obvious
advantage is that the concentration of the drug can be
measured directly andnoninvasively in the tissue of interest,
rather than by biopsy or by inference from indirect or
surrogate measures. The question being asked might be
relatively simple: Does the drug enter the tissue or can it
be part of a larger project aimed at relating the local tissue
concentration time course to that in plasma or to the admi-
nistered dose?
Drug occupancy studies are asking a related but distinct
question: What is the quantitative relationship between the
dose of drug or its concentration in plasma and the occu-
pancy by that drug of a specific molecular target? Here the
approach is to label the target with a radioligand, that is, a
radiotracer which binds specifically to that target, and to
observe with PET the blocking of the binding of the trace
radioligand when the cold (i.e. nonradiolabelled) drug is
administered at pharmacological doses.
General methodology
Choice of radionuclide and labellability
PET radiotracers are commonly labelled with the short-lived
positron-emitting radionuclides 11C, 18F, 13N and 15O (radio-
active half-lives: 20.4, 110, 10 and 2 min, respectively). For
many biological applications, the time constants for uptake
and turnover of small molecules in brain make 11C and 18F
optimal choices of label with half-lives long enough to be
followed during the course of the PET experiment. Typically
this manifests itself in the use of a labelled autologue of the
drug candidatemeasured over a fewhours, or observing on the
kinetic behaviour of a trace radioligand over a similar time
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Drug Discovery Today: Technologies | Imaging technologies Vol. 2, No. 4 2005
periodduring the course of administration of pharmacological
doses of the drug under study.
PET radionuclides are obtained directly from a cyclotron in
the form of simple molecules or ions (e.g. 11CO2,18F-fluor-
ide). These primary labelling materials are converted into
more useful synthetic reagents to enable their incorporation
into the target drug molecule. Because of the short half-lives
of these radionuclides, the repertoire of available synthetic
chemical manipulations is limited. Thus, not all drug candi-
dates can be labelled. A common example of a successful
labelling strategy for carbon-11 compounds is the use of11CO2 which is subsequently converted to 11C-methyl iodide
by a series of rapid chemical manipulations. This reagent can
be used to alkylate O, S, N and C nucleophiles to produce the
corresponding 11C-methylated product. This methodology is
often successfully utilised for the labelling of N-methyl or O-
methoxy containing compounds. For drug molecules not
containing these moieties, alternative labelling approaches
are required. Other labelling reagents available for rapid
labelling include 11C-cyanide, 11C-nitromethane, 11C-phos-
gene and 11C-carbon monoxide. Continuing development of
new synthetic reagents and reactions with radionuclides is of
crucial importance for increasing the ability to implement
successful labelling strategies for PET studies [1].
It is usually the case in drug occupancy studies that the
radioligand has a different molecular structure than the drug.
Indeed some properties of an ‘ideal’ radiotracer are different
from those of the drug, for example, the drug might interact
with several targets but the occupancy study would hope to
answer questions regarding a specific target defined by the
radioligand. Self-occupancy studies, when the drug and the
radioligand are of the same molecular species, do however
have a role to play, particularly in the development of PET
tracers.
Radiolabelled metabolites
PET measures the time course of the total concentration of
radioactivity in the tissue. In the case of CNS studies, if care is
taken in the design of the labelling position in the tracer
molecule, radioactive metabolites generated in the periphery
can be sufficiently hydrophilic to be excluded from tissue
penetration by the blood brain barrier (BBB). Knowledge of
the metabolism of the parent molecule is essential for label-
ling the PET probe in a position whereby brain penetrant
metabolites can be minimised. The fraction of radiolabelled
metabolites can be assayed directly in plasma samples to
enable a proper definition of a parent input function for
kinetic analyses, but because of short half-lives these data
might be limited. Accurate assessment of the contribution of
radiolabelled metabolites in plasma and in the tissue to the
total PET signal is crucial, both in biodistribution and occu-
pancy studies, if the data from a PET experiment are to be
interpreted accurately.
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Kinetic analysis
Mathematical modelling techniques play an essential role in
the interpretation of kinetic PET data [2]. The tissue signal is
dependent on the interrelated processes of tracer delivery in
the blood, transport across the BBB and exchange between
tissue compartments. For radioligands, it is also necessary to
distinguish the free and nonspecifically bound components
from those specifically bound to the target molecule or
receptor. For drug occupancy studies it is usually the last of
these compartments which is of interest. It is important to
emphasise that it is only under special conditions that there is
a simple linear relationship between the tissue/plasma con-
centration ratio and the size of the specifically bound pool,
and in general this is not the case. It would, for example,
require that the concentration of the radioligand is main-
tained constant and at a true equilibrium in both plasma and
tissue. Under conditions where the tracer is administered as
an intravenous bolus, a full kinetic analysis, based on a
validated model, is required to distinguish the components
involved in the total tissue signal and to obtain estimates of
the sizes of the component compartments and the flux
between them.
Labelling the drug: biodistribution studies
In the development of potentially centrally acting drugs, a
basic question is whether the drug crosses the BBB. It is well
recognised that predicting blood–brain permeability for a
given chemical structure is currently very difficult, and there
might be species differences which further confound the
extrapolation from animal studies to human. The adminis-
tration of trace quantities of a radiolabelled drug and its direct
assay in the tissue of interest, as is possible with PET, is often
referred to as ‘microdosing’ and has obvious advantages in
early drug development when the toxicology and pharma-
cology of the drug are less well defined.
Assessment of brain entry
Measurement of the concentration time courses of the radi-
olabelled drug in plasma and in brain allows quantitative
estimation of plasma-to-brain clearance (ml plasma per min
per ml of tissue, K1, equal to the product of flow and extrac-
tion). The data might also allow estimation of an equilibrium
partition coefficient between tissue and plasma. The regional
PET signal contains contributions from the tissue and from a
blood volume component (�5%, v/v). A tracer kinetic model
can be applied to the dynamic PET data, using the plasma
radiotracer concentration as the input function and includ-
ing a blood volume term to allow for the concentration of
tracer in whole blood, to estimate the uptake parameters. For
tracers exhibiting low tissue uptake it is particularly impor-
tant to account for the contribution that the blood volume
and other factors such as out-of-field scatter, make to the PET
signal. A supplementarymeasurement of blood volume based
Vol. 2, No. 4 2005 Drug Discovery Today: Technologies | Imaging technologies
on a radiolabelled carbonmonoxide scan can allow for amore
accurate correction. Interpretation of these parameters must
however take into account the limits of detection.
Nonlinear effects of dosing
In a microdosing study it might be the case that there is no
quantifiable brain uptake of the radioligand, that is, the
radiotracer might not be able to cross the BBB. It could
however be possible that the radiotracer is a substrate for
one or more of the drug efflux transporters such as P-glyco-
protein (Pgp),multidrug resistance proteins or organic anion
transporting polypeptides. These transporters are located in
the endothelial cells of the brain capillaries and keep a tight
regulation on the influx of endogenous and exogenous
agents into the CNS. If it is suspected that the radiotracer
is a substrate for oneof the efflux transporters, further studies
can be performed to assess this phenomenon, for example,
following a higher pharmacological dose of the cold drug,
when saturation of the Pgp might allow uptake of the radio-
tracer. For amore detailed review of the application of PET in
studying the drug efflux transporters in vivo see Loscher and
Potschka [3]. Conversely, if uptake of the radiotracer
involves saturable processes then the fractional uptake of
the radiotracer in a microdosing study will be higher than
that of the cold drug at pharmacological doses. Similar
considerations of nonlinearity apply to the metabolism of
the parent tracer. In certain cases, the metabolism of the
parentmolecule is rapid at tracer doses, whereas for the same
tracer, the situation can be reversed if administered at a
pharmacological dose.
Labelling the target: occupancy studies
In many studies, compounds labelled with positron-emitting
isotopes have previously been characterised for a range of
properties including their selectivity and affinity for target
proteins of interest. This information allows the design of PET
studies that aim to answer specific questions relating to the
characterisation of particular target proteins. Therefore, if a
compound has previously been demonstrated to possess a
high affinity and selectivity for one particular target, then an
occupancy study can be designed where this compound is
labelled with a positron-emitting isotope and administered at
nonpharmacological (trace) doses before and after increasing
pharmacological doses of a structurally dissimilar compound
(which also has an affinity for the target protein of interest).
In studies such as these where the radioligand only labels one
target protein, drugs with an affinity for more than one
protein can be utilised to demonstrate occupancy of the
target protein. The unlabelled compoundmust have a reason-
ably high affinity for the target protein of interest and any
affinity for other proteins must not interfere with the PET
signal, either directly or indirectly. Occupancy studies can
also be designed with radioligands that bind to more than
one target protein. In cases like these, a careful study design
needs to be adopted which will allow for the full block of the
proteins not of interest, followed by assessment of the target
proteins of interest.
Radioligands
Our understanding of small molecule–receptor/tissue inter-
actions is incomplete. Many of the assumptions we use in the
selection and design of radioligands are at best based on a
crude understanding of a very complicated in vivo system.
Successful radioligand development for in vivo imaging is at
present not an exact science, and further advances are
required in understanding small molecule-protein interac-
tions to ensure more efficient selection and design of PET
radioligands in future. In the selection and design of radi-
oligand probes for in vivo imaging, several desirable and/or
essential molecular attributes need to be considered. Two
important factors are lipophilicity and affinity.
In general, successful radioligands are characterised by a
‘window of lipophilicity’: They have sufficient lipophilicity
that they are able to diffuse across biological membranes, but
not so lipophilic that their concentration in lipid-rich tissue
obscures the binding of the radioligand at the molecular
target of interest [4]. In practical terms, several measures have
been used to establish this optimum lipophilicity window,
themost commonly used being the logarithmof the partition
of a molecule between octanol and water (often termed log P,
or log D if performed with the aqueous phase at pH 7.4).
Typically, successful radioligands for CNS applications have
measured log P values in the range 1.5–2.5.
The affinity of a radioligand should be compatible with the
known concentration of the molecular target under investi-
gation. In practical terms,molecular targets expressed in high
concentrations (e.g. dopamine transporter in striatum), can
be delineated with a radioligand with a lower affinity (10–
30 nM) than that required for a target which is present in low
concentrations in tissue (e.g. D2 receptors in cortex require a
radioligand with sub-nanomolar affinity).
Estimation of receptor availability: binding potential
The three-tissue compartment neuroreceptor binding model
described by Mintun et al. [5] has provided a basis for the
kinetic analysis of radioligand studies in PET (e.g. see Ref. [6]).
Thismodel distinguishes three states of the radioligand – free,
nonspecifically bound and specifically bound. The ratio of
the concentration of the radioligand in the specifically bound
state over that of the free state which would occur at tracer
equilibrium is termed the binding potential (BP) and can be
shown, in the simplest case, to equal the ratio of the con-
centration of available receptor sites to the dissociation con-
stant of the radioligand for the receptor (Bavail/KD). Whereas
this is an equilibrium concept, it can be estimated from
analysis of the kinetic behaviour of the radioligand in a
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dynamic PET scan. The BP of the radioligand is a function of
the concentrations and affinities of competing cold ligands,
either endogenous or exogenous, by virtue of their effect on
Bavail. The concentrations of these cold ligands might be in or
near to a steady state and it is the kinetic behaviour of the
radioligand on this background which is of interest. It is the
changes in the BP of the radioligand which indicate occu-
pancy of the receptor sites by the cold drug. In this brief
account we will avoid the use of the loose term ‘radioligand
displacement studies’ because it is potentially misleading,
and has been used incorrectly, for example, to suggest that
a radioligand with a very high affinity is unsuitable for
estimating site occupancy by a cold drug with a relatively
lower affinity because it would not be displaced to the same
extent as a lower affinity radioligand. Consideration of the
above shows this is not the case.
Often, with typical PET data, the individual (micro) rate
constants in the standard PET neuroreceptor binding model
are not well defined andmight be numerically unidentifiable.
However, so-called macro parameters such as K1 (the unidir-
ectional clearance of tracer from plasma to tissue), and the
equilibrium partition coefficient for the total tissue/plasma
concentration ratio, are relatively well defined. (In PET lit-
erature the latter is frequently referred to as a total volume of
distribution, VDtot, in contrast to the use of this term in other
contexts.)
The existence of a reference region for the radioligand, that
is, a region of brain where there is negligible specific binding,
when this occurs, is of particular value if it can be assumed
that the size of the free and nonspecific compartments is the
same in reference and target tissues. If an arterial plasma
parent input function is available (the ideal case) then a
comparison of VDtot in reference and target allows estimates
of binding potential, BP1, to be obtained (BP1 = f1Bavail/KD,
where f1 is the fraction of the tracer in plasma which is free
and unbound to plasma protein, lipid among others; Bavail is
the concentration of available receptor sites in the tissue, and
KD the apparent equilibrium dissociation constant for the
ligand), together with BP2, the corresponding expression
incorporating the free fraction in the tissue, f2. A second
important consequence of the existence of a reference region
is the possibility of avoiding arterial blood sampling alto-
gether and expressing the target tissue kinetics as a function
of those of the reference tissue [2]. This allows estimates only
of BP2. In all these cases, however, the obvious points should
be emphasised, including the fact that in a tracer-alone
experiment, changes in Bavail and KD cannot be distinguished
and that changes in BP can also reflect changes in free
fractions. However, if these assumptions are clear and satis-
factory, the way is open for the very powerful techniques of
estimating receptor occupancy by a drug, either for the
purpose of simply demonstrating that the drug has access
to the site when direct biodistribution data cannot be
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obtained or for establishing a quantitative relationship
between dose and occupancy.
Brain penetration and receptor occupancy at pharmacological doses
As a simple example here, let us consider a case of a baseline
scan (radioligand alone) together with scans carried out with
three increasing doses of the cold drug under study. Let us
assume that these scans are carried out with no allowance for
interim analysis and adaptive design of doses. Let us also
assume a simple single-site competition model. The actual
range of the three doses will depend on the question being
asked; however, the key factor in determining the doses will
be an initial estimate of the in vivo IC50 for the compound
under investigation. This could be based on allometric scaling
[7] or extrapolation from in vitro studies. So, for the two
questions which are typically asked:
� C
NS penetration: Here the goal is to investigate thehypothesis that the cold drug gets into the brain. Given
the focus of the question it is important to settle for a high
enough dose to avoid any false negative (Type II error).
Thus, a reasonable scheme for dose selection in our simple
example is the three doses which are D/10, D, 10D (subject
to consideration of themaximum tolerable dose). A reason-
able scaling of this range is to choose D to be the estimated
IC50. If this estimate is correct and the compound is CNS
penetrant, this should lead to occupancy values of 9%, 50%
and 91%. However, based on the need to avoid a Type II
error and one’s confidence in the predicted IC50, itmight be
appropriate to choose D to be greater than the estimated
IC50. So, themain requirement is to have an estimate of the
IC50 and some confidence interval for this estimate.
� D
ose–occupancy curve: Here the goal is to obtain a doseoccupancy curve and measure the IC50 of the compound
directly. For such studies one might employ a tighter range
of doses than for a CNS penetration study, given that an
accurate estimate of the IC50 is required. A possible strategy
is to aim for 25%, 50% and 75% occupancy based on doses
of D/3, D, 3D (subject to maximum tolerable dose). How-
ever, it might be prudent to alter the range of doses
depending on one’s confidence in the estimate of the
IC50 (=D), but again an optimal design centres on having
an initial estimate of the IC50.
It is important to note here that even if one is interested in
establishing a dose of drug with a particular target occupancy
inmind, it is better to attempt to define the whole occupancy
curve from low to high. A standard receptor-binding model
can then be used to interpolate or extrapolate to the desired
occupancy, circumventing signal-to-noise problems at high
and low occupancies. The above simple examples are for
illustrative purposes. Then, for studies involving more
scans/subjects, one can use more intelligent adaptive designs
Vol. 2, No. 4 2005 Drug Discovery Today: Technologies | Imaging technologies
in which the subsequent doses that are used can be altered
depending on values obtained from previous scans.
Thus, it is clear that PET occupancy studies can help define
an optimal dose, particularly if the target occupancy is pre-
defined. However, it goes without saying that extrapolations
from PETmeasures to optimal dose are heavily dependent on
assumptions regarding the mode of action of the drug and its
relationship to the disease process, and that it is necessary to
be very clear about what constitutes ‘optimal’. It might be
useful, when considering the contributions that PET studies
could make to the establishment of an optimal dosing regi-
men to consider the PET study as providing a ‘snapshot’, in
time, of the availability of the drug at its site of action. The
bioavailability of the drug under study, usually administered
orally, is a function of its absorption and whole body meta-
bolism and excretion. The PET tracer is usually administered
intravenously and gives a tissue response function relative to
plasma, at specific times, during this period. An early example
is the study by Bench et al. [8] looking at the clearance of a
drug from a receptor following a study in which a dose–
response curve had been established. Here we are merely
concerned with some specific and basic methodological con-
siderations to be taken into account for the type of PET
studies outlined above.
Conclusion
In this brief overview of PET methodology, we have touched
upon a few selected topics which are by no means compre-
hensive. PET is a multidisciplinary modality in Experimental
Medicine, relying on contributions from Physics, Chemistry,
Mathematics and Biology.
Advances which will impact on the field will undoubtedly
come from further developments in chemistry, in particular a
better understanding of the relationship between properties
of compounds in silico and their behaviour as tracers in vivo. In
addition to the pharmacology associated with specifically
bound state of the tracer, its behaviour in its free and non-
specifically bound states is equally important. The free frac-
tion of the tracer in tissue is amajor factor in determining the
suitability of a compound as a PET tracer. The further devel-
opment and validation of reference tissue modelling is cru-
cially dependent on a better understanding in this area. In
addition, more knowledge of factors affecting the free
fraction of the tracer in plasma and improvements in the
methodology of assaying this in individual subjects is
required, not only for tracer design but also for more accurate
quantitation of the PET signal.
We have emphasised the necessity for appropriate model-
ling to take full advantage of the quantitative PET signal.
Although there is a growing body of accepted methodologies
for quantitative analysis, it is important to carefully examine
the assumptions and limitations of these methods in each
individual case and not lose sight of the underlying biology
and pharmacology. It is the quantitative nature of the data
and their analysis that will allow for appropriate objective
decisions to be made during the drug development process.
Here we have concentrated on particular current applica-
tions, namely biodistribution and occupancy studies in brain.
There is also an increasing interest in applications of similar
techniques in other tissues. Such studies are being employed
to accelerate drug discovery and development, and are of
great value, enabling noninvasive measurements in humans
early in the drug development pipeline. There aremany other
potential applications of PET that could ultimately impact on
this area. These include techniques for imaging gene expres-
sion, characterising the downstream response to drugs and
the characterisation of novel metabolic pathways.
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