1. health and fitness
2. disease

Research Overview
Highlights of PET Studies on Chiral Radiotracers and Drugs
at Brookhaven
1
DDR
DRUG DEVELOPMENT RESEARCH 59:227–239 (2003)
Yu-Shin Ding1,2n and Joanna S. Fowler1
Chemistry Department, Brookhaven National Laboratory, Upton, New York
2
Medical Department, Brookhaven National Laboratory, Upton, New York
Strategy, Management and Health Policy
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Preclinical Development
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Drug Delivery,
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Phases I-III
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Phase IV
ABSTRACT
We review several PET studies of chiral molecules which have been carried out in our
laboratory. In many cases the enantiomers behave differently, reflecting factors such as differential
specificity for enzymes and transporters and binding to plasma proteins, as well as selective binding to
receptors. These studies demonstrate that PET imaging is a suitable method to investigate the behavior of a
chiral drug in the human body and is a powerful tool in drug development. It is important to emphasize the
essential and pivotal role that organic synthesis has played in the development of PET during the last
quarter century. PET is now an important tool in the neurosciences, cardiology, and oncology and is
available in hundreds of institutions worldwide. However, PET is by no means mature in terms of the study
of chiral drugs; hundreds of racemic drugs have been developed but only a few have been labeled with
positron emitters to study their stereoselectivity. One reason is the complexity of rapid synthesis, including
chiral synthesis and chiral purification. New developments in these areas are needed. Although PET is a
challenging and expensive technology, it is exquisitely suited to functional and neurochemical studies of
the human brain and other organs. Its use in drug research and development holds promise in
understanding duration of action and stereoselectivity of drugs and in facilitating drug discovery and the
c 2003 Wileyintroduction of new drugs into the practice of medicine. Drug Dev. Res. 59:227–239, 2003. Liss, Inc.
Key words: positron emission tomography; chiral drugs; carbon-11; fluorine-18; stereoselectivity
INTRODUCTION
Positron emission tomography (PET) is a medical
imaging method which uses radiotracers labeled with
short-lived positron-emitting isotopes, such as carbon11 (half-life 20 min) and fluorine-18 (half-life 110 min)
to track biochemical transformations, changes brought
about by disease, as well as the movement of drugs in
the living human and animal body. The use of PET to
examine drug pharmacokinetics and pharmacodynamics and the relationship of these properties to
the behavioral, therapeutic, and toxic properties of
drugs is emerging as a powerful new scientific tool.
PET provides a new perspective on drug research by
virtue of its ability to directly assess different parameters such as absolute uptake, regional distribution
and kinetics, bioavailability, drug binding mechanism,
c 2003 Wiley-Liss, Inc.
duration of action, and metabolic effects in human
subjects. For example, the labeled drug itself can be
used to measure the absolute uptake, regional distribution, and kinetics at its site of action in the body.
Additionally, the labeled drug and whole-body PET can
be used to determine the target organs for the drug and
its labeled metabolites and thus provide information on
potential toxic effects as well as tissue half-lives. On the
other hand, different labeled tracers can be used to
n
Correspondence to: Yu-Shin Ding, Chemistry Department,
Brookhaven National Laboratory, Upton, NY 11973-5000.
E-mail: [email protected]
Published online in Wiley InterScience (www.interscience.
wiley.com) DOI: 10.1002/ddr.10221
228
DING AND FOWLER
assess the effects of a drug on particular physiological
or functional processes. For example, with appropriate
radiotracers the effects of a drug on metabolism,
neurotransmitter activity, blood flow, enzyme activity,
or other processes can be probed. Because of the short
half-life of the positron emitters, these parameters
can be assessed directly in the human body both in
normal controls and in patients and series studies
can be done where a subject serves as his/her own
control. Moreover, multiple tracers can be used so that
different parameters can be assessed in the same
subject. An important point is that PET can be used
to assess the behavior of a drug at its site of action
directly in human subjects. This is relevant because
drug pharmacokinetics and availability may vary across
animal species. It also enables the assessment of
drug behavior in diseases where there are no animal
models. This information is not available with other
methods and places PET in a unique position to
streamline the process of drug development and
evaluation. PET has also found increased application
in studies that examine the behavior of chiral drugs in
the human body and is a valuable tool in drug
development.
To date, clinical studies have shown that it is most
beneficial to use single-enantiomer drugs when one of
the isomers proves to be toxic or causes undesirable
side effects; for example, many of the serious side
effects encountered with racemic dihydroxyphenylalanine (D,L-DOPA), such as granulocytopenia, were not
seen with L-dopa [Thall, 1996]. In another example,
R-(–)-methadone, the more active component of
racemic methadone which is used for the treatment
of heroin addiction, is prescribed to patients suffering
from severe liver damage in order to avoid the extra
burden of metabolizing the inactive enantiomer [Kreek
et al., 1979]. That is, if the beneficial effects of the drug
reside only in one enantiomer of the racemic drug,
then 50% of the weight of the administered drug may
not contribute to its therapeutic effects, or may interact
to influence the behavior of the active form by multiple
mechanisms, or it may contribute to its side effects. It is
therefore not surprising to find a steady, rising flow of
single-isomer forms of chiral drugs into U.S. and world
markets; the annual sales of enantiomeric drugs is set
to break through the $100 billion mark in 2000. This
creates an increasing demand for enantiomeric intermediates and bulk active compounds as well as
enantioselective technology and services. With the
current trend in the pharmaceutical industry to
develop optically pure products, the stereoselectivity
of drugs has become a timely subject. A noninvasive
imaging method such as PET to determine or
discriminate the behavior of individual enantiomers
in the living system is therefore crucial in drug
development and research.
In this report, we describe highlights of PET
studies on chiral drugs at the Brookhaven National
Laboratory. We do not intend to review all our PET
studies with chiral molecules, such as L-ornithine
[Ding et al., 1989], (–)-50 -18F-D8-THC [Charalambous
et al., 1991], or (7)norchlorofluoroepibatidine [Ding
et al., 1999], but will focus on comparative PET studies
with enantiomers of various labeled drugs and radiotracers in the fields of neuroscience (brain and heart)
and neuropharmacology.
(–) AND (+)-[18F]BMY 14802, A POTENTIAL
ANTIPSYCHOTIC DRUG
BMY 14802 (a-(4-fluorophenyl)-4-(5-fluoro-2pyrimidinyl)-1-piperazinebutanol) is a fluorine-containing compound developed as an antipsychotic drug.
Initial studies with BMY 14802 in vitro and in animals
predicted that it would have antipsychotic properties
without the extrapyramidal effects accompanying most
conventional antipsychotic medications [Taylor and
Dekleva, 1988]. For example, BMY 14802 prevents
the behavioral effects of coadministered amphetamine
and does not cause catalepsy [Taylor and Schlemmer,
1992]. It was also predicted that BMY 14802 would be
more effective in treating the negative symptoms
of schizophrenia than the typical antipsychotic drugs.
BMY 14802 does not have the pharmacological profile
of typical antipsychotic drugs which have a common
characteristic of binding to the dopamine D2 receptors.
BMY 14802 binds in vitro with moderate affinity
for sigma binding sites but has very low affinity
for dopamine D2 receptors (IC50 ¼ 112 nM for
(7)BMY 14802 for inhibition of binding of a sigma
radioligand [3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP), and IC50 ¼ 6,400 nM for inhibition
of binding of a dopamine D2 radioligand [3H]spiperone) [Taylor and Dekleva, 1988]. Although BMY 14802
is being evaluated for clinical use as the racemic
mixture, it has a chiral center with the (+)-enantiomer
having a 10-fold higher potency for the sigma receptor
than the (–)-enantiomer (IC50 ¼ 28 nM for the (+)enantiomer and IC50 ¼ 310 nM for (–)-enantiomer
against [3H]3-PPP) [Taylor et al., 1991]. In order to
assess the brain uptake, pharmacokinetics, stereoselectivity, and binding properties of this potential antipsychotic drug, enantiomerically pure samples of (–)
and (+)-[18F]BMY 14802 were synthesized and examined in baboon with PET (Fig. 1).
The labeled racemic form of the drug ([18F]-(7)BMY 14802) was used for most of these studies since
this is the form targeted for safety and clinical trials.
However, to probe selective binding to the sigma
HIGHLIGHTS OF BROOKHAVEN PET STUDIES
229
Fig. 1. Synthesis and resolution of (7)-[18F]BMY 14802.
receptor, a comparative PET study was also carried out
with the (+)-enantiomer, which has a 10-fold higher
affinity for the sigma receptor. The peak uptake of
[18F]BMY 14802 in the brain was high (0.04–0.07% of
the injected dose/cc in the brain at about 5 min, or
about 8–14% of the injected dose), reflecting a high
penetration of the drug across the blood–brain barrier,
consistent with its partition coefficient of 55 at pH 6.7,
which is optimal for the diffusion of molecules across
the blood–brain barrier [Goldstein and Betz, 1986].
However, in spite of a large initial delivery of tracer to
the brain, clearance from all regions of the brain was
very rapid. The radioactivity cleared to about 30% of
peak value by 20 min postinjection and to less than 10%
of the peak value by 60 min postinjection in all regions.
Total radioactivity cleared rapidly from the plasma and
the percentage of free [18F]BMY 14802 fraction
decreased from 95% to 35–50% by 10 min, with a
corresponding increase in the fraction of glucuronidated [18F]BMY 14802.
The regional distribution was also examined from
the perspective of the distribution volume (DV), which
is a function of receptor concentration (Bmax), since
regions with a greater concentration of sigma binding
sites would be expected to have larger DVs. However,
the distribution of relative values of the DVs within a
baboon does not parallel the known regional distribution of sigma sites (cerebellum 4 frontal cortex 4
striatum 4 thalamus) measured in postmortem brain
tissue [Walker et al., 1990; Weissman et al., 1988].
Furthermore, there was no expected reduction in DV
after pretreatment with 7 mg/kg of unlabeled BMY
14802. These results suggest that if BMY 14802 is
binding to sigma or other receptors, then specific
binding (i.e., Bmax/Kd) is small compared to nonspecific
binding. This could be due to a rapid dissociation of the
ligand from the receptor, making Kd significantly larger
than Bmax. Therefore, the observed DV in different
regions of the brain is due primarily to variations in the
ratio of transport constants K1/k2 which are large
enough to mask variations in Bmax/Kd (if receptor sites
are involved). Thus, the behavior of BMY 14802
appears to be dominated by transport properties of
the drug rather than by its binding to sigma receptors.
This is supported by comparative studies with labeled
racemic and (+)-BMY 14802 (the enantiomer with
higher affinity for the sigma binding site), where no
significant differences were observed (Fig. 2).
In the initial development of a therapeutic drug,
the in vitro pharmacological profile is frequently used
to guide the selection of priority structures for further
testing. However, while a promising pharmacological
profile measured in vitro is a relatively rapid and
effective initial assessment of the potential for therapeutic efficacy in vivo, it gives no indication of the
delivery of the drug to pharmacologically relevant sites
in vivo. In vivo, factors such as rapid metabolism, high
binding to plasma proteins, poor penetration of the
blood–brain barrier, or rapid brain clearance can limit
the concentration of a drug at its pharmacologically
relevant site. While poor retention at the target site
may be compensated for by the administration of large
doses of the drug, considerations of drug toxicity are
usually a limiting factor. During the time when these
PET studies of [18F]BMY 14802 were being carried
out, safety trials with BMY 14802 were carried out in
schizophrenic subjects. BMY 14802 was administered
in doses of 500 mg four times a day for 22 days with no
adverse effects. Thus, in spite of the rapid clearance of
BMY 14802, a reasonably high concentration of the
230
DING AND FOWLER
This study illustrates the value of using PET in
the early phases in the development of a new drug to
assess its pharmacokinetics and to reveal potential
problems in achieving effective concentrations at
pharmacologically relevant sites which are not predicted with in vitro binding profiles.
ENZYMES AS MOLECULAR TARGETS IN DRUG
DEVELOPMENT: STUDIES OF BRAIN MAO B USING
(R)-(–)- AND (S)-(+)-[N-11C-METHYL]-DEPRENYL,
AND (R)-(–)- AND (S)-(+)-[N-11C-METHYL]4FLUORODEPRENYL
Fig. 2. Time–activity curve for (7)-[18F]BMY 14802 (squares) vs. (+)[18F]BMY 14802 (circles) in cerebellum.
drug might be obtained in the brain because the drug is
well tolerated at very large doses. However, the
ultimate determinant of the therapeutic effectiveness
of a centrally acting drug is whether it is associated with
pharmacologically relevant sites in the brain. Interestingly, safety trials of BMY 14802 in schizophrenic
patients receiving 3,000 mg/day carried out while this
PET study was in progress showed disappointing levels
of efficacy. Whether this is due to its rapid clearance
from brain and the resulting inability to achieve a
pharmacologically effective concentration of the drug is
not known.
This study raises an important issue in drug
research and development, namely, the relationship
between the concentration and residence time of a
drug in the brain and its pharmacological effects. The
results of these PET studies with [18F]BMY 14802
make it tempting to speculate that rapid clearance and
failure to measurably associate with sigma binding sites
in vivo could account for initially disappointing clinical
trials in humans. However, there are a number of
examples of drugs which rapidly clear from brain but
which have measurable pharmacological effects over a
much longer time such as tetrabenazine [DaSilva et al.,
1992; Pletscher et al., 1962]. There are also an
increasing number of examples where the administration of a single dose of certain drugs such as
amphetamine can produce long-term effects [Antelman et al., 1991]. Thus, in the absence of other
knowledge concerning the binding profile of a drug and
its potential indirect actions, one cannot yet make
general predictions regarding the concentrations and
residence times required to achieve pharmacological
effects.
Enzymes are common molecular targets in drug
development [Hansch et al., 1990; Palfreyman et al.,
1989]. Thus, the development of PET tracers which
can be used to assess the effects of a drug on enzyme
activity at the site of action of the drug can be of
potentially great importance in drug development.
2-Deoxy-2-[18F]fluoro-D-glucose (FDG) is the most
widely used PET tracer and is metabolically trapped by
its reaction with the glycolytic enzyme hexokinase
[Reivich et al., 1979]. It has been used extensively in
the study of therapeutic drugs and substances of abuse
[Fowler et al., 1990]. Ornithine decarboxylase (ODC)
is known to be expressed early in the deregulation of
cellular activity and is one of the markers for rapid
tissue growth; it therefore has been a target for
chemotherapeutic drugs. It has also been the target
for tracers assessing increases in ODC activity. One of
these is L-5-11C]ornithine [Ding et al., 1989]. ODC
catalyzed decarboxylation of L-5-[11C]ornithine would
produce 1-[11C]putrescine which would be retained at
the site of production. Note that labeling ornithine at
the carboxyl carbon would result in a loss of label at the
decarboxylation step. The tumor uptake of D,L-5[14C]ornithine and a comparative study of ODC activity
in tumor with L-1-[14C] and L-5-[14C]ornithine have
been reported, thus supporting this approach [Elsinga
et al., 1989; Ishiwata et al., 1988].
Monoamine oxidase (MAO) is another important
enzyme which is responsible for metabolizing endogenous neurotransmitter amines. It occurs as two
subtypes, MAO A and MAO B, on the basis of
substrate and inhibitor selectivity. Interest in MAO as
a therapeutic target was stimulated in the early 1950s,
when it was discovered that the inhibition of MAO
produced antidepressant effects. L-Deprenyl ((R)-(–)deprenyl), a selective irreversible MAO B inhibitor, was
found effective in combination with L-DOPA therapy
in Parkinson’s disease to inhibit the degradation of
dopamine. In order to study the functional MAO
activity in the living brain, we have synthesized C-11labeled L-deprenyl and carried out PET studies in vivo
in animals [Arnett et al., 1987; MacGregor et al., 1985]
HIGHLIGHTS OF BROOKHAVEN PET STUDIES
and in humans [Fowler et al., 1987]. The results
showed that [11C]L-deprenyl localized in MAO B-rich
regions (striatum, thalamus, brainstem) and that the
labeled products are effectively trapped over a 90-min
time interval and that the recovery of MAO B after
irreversible inhibition has a 6-week half-life in human
brain [Fowler et al., 1994a].
Deprenyl contains an asymmetric carbon and the
MAO inhibitory potency of the L-enantiomer of
deprenyl has been shown to exceed that of the Denantiomer B25 times [Robinson, 1985]. In order to
assess the stereoselectivity of the drug in the human
brain and to support the notion that binding of
deprenyl in the brain involves MAO B, a comparison
study of [11C]L- and [11C]D-deprenyl was carried out
with PET. The results demonstrated rapid clearance of
the inactive (D-) and retention of the active (L-)
enantiomer within MAO B-rich brain structures, in
agreement with the known stereoselectivity (Fig. 3). In
addition, mechanistic PET studies using deuteriumsubstituted [11C]L-deprenyl have identified catalysis by
MAO as the rate-limiting step in the retention of
radioactivity in the brain after the injection of the
tracer [Fowler et al., 1988, 1995].
As part of our interest in the development of a
fluorine-18 labeled derivative of L-deprenyl,
we assessed the effect of fluorine substitution on
deprenyl by synthesizing (R)-(–)- and (S)-(+)- and
(R,S)-(7)-[N-11C-methyl]4-fluorodeprenyl and comparing their regional uptake in baboon brain by using
PET [Plenevaux et al., 1990a,b]. As shown in
Figure 4, (R)-(–)- and (S)-(+)-4-fluorodeprenyl were
synthesized via the reaction of 4-fluorobenzaldehyde
Fig. 3. Comparative studies of L-[11C]deprenyl in human brain: time
course in thalamus before (open symbols) and after (solid symbols) a
15 mg dose of unlabeled L-deprenyl.
231
with nitroethane followed by reduction with lithium
aluminum hydride to produce racemic 4-fluoroamphetamine, which was resolved by recrystallization with
L- or D-acetylleucine to yield (R)-(–)- and (S)-(+)-4fluoroamphetamine in 496% enantiomeric excess with
yields of 42% and 39%, respectively. Alkylation with
propargyl bromide gave (R)-(–)- and (S)-(+)-4-fluoronordeprenyl which was reductively methylated to
produce (R)-(–)- and (S)-(+)-4-fluorodeprenyl. Alkylation of (R)-(–)- and (S)-(+)-4-fluoronordeprenyl with
[11C]methyliodide afforded (R)-(–)- and (S)-(+)[N-11C-methyl]4-fluorodeprenyl in a radiochemical
yield of 30–40%.
The distribution of radioactivity for the three
tracers (R)-(–)-, (S)-(+)-, and (R,S)-(7)-[N-11Cmethyl]4-fluorodeprenyl) in the striatum, thalamus,
and cerebellum in the baboon brain were markedly
different. The absolute striatal uptake at 60 min
was 0.053% injected radioactivity per cc for
(R)-(–)-, 0.021% for (S)-(+)-, and 0.041 for (R,S)-(7)[11C]4-fluorodeprenyl. The value for (R)-(–)-[11C]4fluorodeprenyl was very similar to that of (R)-(–)[11C]deprenyl itself (0.057% injected radioactivity per
cc) [Plenevaux et al., 1990a,b]. These studies suggest
Fig. 4. Synthesis of (R)-(–)- and (S)-(+)-4-fluorodeprenyl and (R)-(–)and (S)-(+)-[N-11C-methyl]-4-fluorodeprenyl.
232
DING AND FOWLER
that (R)-(–)-[11C]4-fluorodeprenyl would be a good
tracer for PET studies of MAO B.
COMPARATIVE PET STUDIES OF (+)-[11C]COCAINE
AND (–)-[11C]COCAINE
Of the two enantiomers of cocaine, only one,
(–)-cocaine, is found in Erythroxylon coca leaves. It has
the 1R, 2S, 3S, 5S configuration [Hardegger and Ott,
1955]. Although it is reported that synthetic
(+)-cocaine can act as an inhibitor, it is much weaker
than (–)-cocaine for presynaptic monoamine uptake
[Ritz et al., 1987], and lacks the behavioral properties
of the levorotatory isomer [Spealman et al., 1983]. We
have labeled the naturally occurring enantiomer of
cocaine, (–)-cocaine, with C-11 on the N-methyl group
and showed that cocaine is rapidly taken up in the
striatum of human brain with a time course similar to
that of the subjective behavioral ‘‘high’’ [Fowler et al.,
1989]. We also prepared (+)-[11C]cocaine with an aim
of using the unnatural enantiomer to better define the
relative contributions of specific and nonspecific
binding of [11C]cocaine to the brain uptake.
When (+)-[11C]cocaine was administered to
baboon, the amount of 11C that entered the brain
was below the detectable limits of PET, contrasting
markedly with the high uptake in the basal ganglia after
injection of (–)-[11C]cocaine (Fig. 5). The time course
of 11C from (+) and (–)-[11C]cocaine in the basal
ganglia confirms the virtual absence of label in the
brain for the (+)-compound. This profound difference
in brain uptake was unexpected, because cocaine,
which has an octanol/water partition coefficient of 7.6,
should have a single-pass extraction fraction across the
blood–brain barrier of near 1 [Goldstein and Betz,
1986]. The explanation for the lack of uptake was
Fig. 5. Time-activity curves for [11C](+)-cocaine (squares) and
[11C]()-cocaine (circles) in striatum.
determined to be very rapid metabolism of (+)-cocaine
in the blood. By 30 sec after administration of (+)[11C]cocaine, it was undetectable in plasma. In vitro
studies demonstrated that (+)-cocaine is 50% debenzoylated to (+)-ecgonine methyl ester with 5 sec of
exposure to baboon plasma but not to washed
erythrocytes. Serum butyrylcholinesterase (BChE; EC
3.1.1.8) appears to be responsible for this hydrolysis
[Gatley et al., 1990]. (+)-Cocaine was hydrolyzed by
BChE over 2,000 times faster than naturally occurring
(–)-cocaine. This strongly suggests that the lack of
behavioral effects of (+)-cocaine may have been due in
part to the unnatural enantiomer failing to survive in
the blood long enough to reach the brain. It is also
possible that the binding of (+)-cocaine to dopamine
transporter in brain tissue homogenates may have been
underestimated because the brain contains BChE
[Chatonnet and Lockridge, 1989]. Our results emphasize that, just as for therapeutic drugs, it cannot be
assumed that interaction with the target receptor or
enzyme is the only difference between the in vivo
behaviors of enantiomeric radiopharmaceuticals.
COMPARATIVE PET STUDIES OF [11C]D-THREOMETHYLPHENIDATE AND [11C]L-THREOMETHYLPHENIDATE [11C]D-THREOMETHYLPHENIDATE AS A RADIOTRACER TO STUDY
DOPAMINE TRANSPORT
Methylphenidate (MP, dl-threo-methyl-2-phenyl2-(2-piperidyl)acetate (Ritalin) is the most commonly
prescribed psychoactive medication for children in the
US, where it is used for the treatment of attention
deficit hyperactivity disorder (ADHD) [Barkley, 1977].
It is a chiral drug and is marketed as the racemic
mixture of the d-threo and l-threo forms. The
psychostimulant properties of MP have been linked
to its binding to a site on the dopamine transporter,
resulting in inhibition of dopamine reuptake and
enhanced levels of synaptic dopamine. It is this
stimulation which is believed to regulate attention
and impulsivity of ADHD children. Our first PET
studies of C-11-labeled racemic dl-threo-methylphenidate ([11C]MP) in the baboon and human brain
demonstrated the saturable [11C]MP binding to the
dopamine transporter and its sensitivity to dopamine
neuron degeneration in Parkinson’s disease [Ding et al.,
1994a,b]. The difference in the pharmacokinetics of
[11C]cocaine and [11C]MP in human brain provides
insight concerning their differences in drug properties;
cocaine is a drug of abuse and MP is a drug of choice.
These studies allow us to better understand the
reinforcing ability of psychostimulant drugs [Volkow
et al., 1995]. The comparative studies of oral methylphenidate and intravenous cocaine highlight the
HIGHLIGHTS OF BROOKHAVEN PET STUDIES
233
proval of the labeled drug for human PET studies
through the Radioactive Drug Research Committee
(RDRC) route. The observation of higher specific-tononspecific binding as compared to racemic [11C]MP
and the selectivity to dopamine transporters and
reversibility demonstrates that [11C]d-threo-MP is a
suitable PET radiotracer to probe the neuronal loss
occurring in drug abuse, normal aging, and in
neurodegenerative disease [Wang et al., 1995; Volkow
et al., 1996a–h].
COMPARATIVE STUDIES OF [11C]D- AND L-THREOMP IN HUMAN BRAIN
Fig. 6. Ratios of striatum-to-cerebellum for [11C]d-threo-MP,
[11C]MP, and [11C]l-threo-MP in the same baboon.
importance that route of administration and hence rate
of brain uptake have on the reinforcing effects [Volkow
et al., 1999].
It has been shown that d-threo-MP is more
potent in the induction of locomotor activity and has a
higher affinity for the dopamine transporter than
l-threo-MP [Barkley, 1977; Pan et al., 1994; Patrick
et al., 1987; Ritz et al., 1987; Schweri et al., 1985]. With
the intention to develop a new PET ligand for studying
the dopamine transporter in humans, we have
developed the synthesis of enantiomerically pure
C-11-labeled d- and l-MP ([11C]d-threo-MP and
[11C]l-threo-MP). Characterization of the binding of
[11C]d-threo-MP in the baboon and human brain with
PET has shown that its specific binding is predominantly to the dopamine transporter, and that pretreatment with drugs that bind to the dopamine transporter
can completely inhibit its specific binding [Ding et al.,
1995; Volkow et al., 1995]. The specific binding of
[11C]d-threo-MP was higher than that for [11C]MP and
[11C]l-threo-MP (3.3 for d-MP, 2.2 for racemic dl-MP,
and 1.1 for l-MP in the same baboon, Fig. 6). Its
percentage of uptake in brain is similar to that of
[11C]cocaine (7–9%) but its clearance is slower. The
slower clearance of [11C]d-threo-MP is an advantage
for kinetic analysis in that it is fast enough to allow for
proper quantification but is slow enough to permit
appropriate counting statistics. Furthermore, its quantification is not confounded by the presence of l-threoMP (the less active enantiomer). Another advantage for
[11C]d-threo-MP is that, as an approved drug, unlabeled methylphenidate can be given in humans to
assess nonspecific binding, thus facilitating the ap-
ADHD, which is estimated to affect more than 2
million children (3–5% of all children), has become
America’s No. 1 childhood psychiatric disorder and MP
(Ritalin) is the drug of choice for the treatment of
ADHD. It is made up of a mixture of two molecules,
d-threo and l-threo. An important concern is, ‘‘should
we use a racemic drug or single enantiomer?’’ While
therapeutic activity often resides in one enantiomer, the
other can lead to undesirable side effects. For most
chiral drugs, the advantages of using a single enantiomer are evident: smaller doses, products twice as active,
fewer side effects, and superior pharmacological
profiles of the active compound. The ability to label
dl-threo-MP as well as the individual enantiomers,
d-threo and l-threo-MP, with carbon-11 provided the
opportunity to compare them directly in the human
brain with PET [Ding et al., 1997]. Figure 8 shows
images of [11C]d-threo-methylphenidate and [11C]lthreo-methylphenidate in the same normal volunteer.
In Figure 7, the time course of the two enantiomers is
compared. [11C]d-threo-MP has a high uptake and
retention in the basal ganglia, the brain region with the
highest concentration of dopamine transporters. In
contrast, [11C]l-threo-MP clears rapidly in both basal
ganglia and cerebellum. Studies in baboons which
had been pretreated with unlabeled MP and other
dopamine transporter blocking drugs showed that
dopamine transporter blockade markedly decreased
the retention of [11C]d-threo-MP in the basal ganglia
but had no effect on the cerebellum. In contrast,
pretreatment with unlabeled methylphenidate had no
effect on [11C]l-threo-MP, indicating that the l-enantiomer shows no specific retention. These results are
consistent with a number of behavioral, in vitro, and ex
vivo studies [Ferris, 1972; Eckerman et al., 1991;
Patrick et al., 1987; Schweri et al., 1985]. Our
microdialysis studies in free-moving rats, which allow
us to directly measure the effect of a drug on
extracellular striatal dopamine in the brain, showed
that d-threo-MP increased extracellular dopamine
concentration by 650%, whereas l-threo-MP did not
234
DING AND FOWLER
affect dopamine levels. These results indicate that
pharmacological specificity of MP resides entirely in the
d-threo isomer and directly show that binding of the lisomer in human brain is mostly nonspecific. This
Fig. 7. Individual time–activity curves in basal ganglia (squares) and
cerebellum (circles) for a human subject after injection of [11C]dthreo-MP (solid symbols) and [11C]l-threo-MP (open symbols). Note
the much higher uptake of [11C]d-threo-MP in the basal ganglia as
compared to that of [11C]l-threo-MP.
Fig. 8. Transaxial PET images (Planes 8, 9, 10, and 12 on an
averaged emission scan representing the activity from 10–90 min) of
the human brain after injection of [11C]d-threo-MP (top panel) and
[11C]l-threo-MP (bottom panel). Images are from the top of the brain to
reemphasizes the very important issue of whether or
not we should use a racemic drug or a single
enantiomer. It is estimated that approximately 1.5 tons
of MP were used in the US in 1990; annual sales of the
drug exceed $350 million. Studies also indicate that
those with untreated ADHD are more likely to become
alcoholics, smokers, or drug abusers than the general
population [Gittelman et al., 1985]. The mechanism(s)
which make MP work for ADHD patients is still
unclear. It has been suggested that the stimulant
appears to increase the level of extracellular dopamine
in the frontal lobe of the brain [Butcher et al., 1991;
Volkow et al., 1994; Zetterstrom et al., 1988] where it
regulates attention and impulsivity. In addition, recent
SPECT studies have shown elevated dopamine transports in the ADHD adult which could contribute to
reduced synaptic dopamine [Dougherty et al., 1999;
Dresel et al., 2000]. The difference in terms of
therapeutic effects and side effects that are involved
by using the d-form alone as compared to the use of the
racemic drug would in principle help us to better
understand the mechanism(s). These studies also
demonstrate that PET imaging is an ideal way to
examine the behavior of a chiral drug in the human
brain and is a valuable tool in drug development.
the base of the skull (left to right). Note the high accumulation of
radioactivity in the basal ganglia for [11C]d-threo-MP as compared to
that for [11C]l-threo-MP.
HIGHLIGHTS OF BROOKHAVEN PET STUDIES
PET STUDIES OF (+)- AND (–)-6[18F]FLUORONOREPINEPHRINE IN THE HEART
(–)-Norepinephrine ((–)-NE) is the principal
neurotransmitter of the mammalian sympathetic nervous system and a major CNS neurotransmitter [Esler
et al., 1990]. Studies with [3H]NE showing that it is
taken up and stored in sympathetic nervous tissue and
released by nerve stimulation have formed the basis for
its use as an index of sympathetic nervous function
[Goldstein et al., 1988]. Accordingly, many studies in
animals and humans have used (–)-[3H]NE as a tracer
to label the endogenous NE pool and to assess changes
in adrenergic activity resulting from disease, stress,
drugs, and other factors by observing the overflow of
labeled norepinephrine and its metabolites into the
plasma [Esler et al., 1990]. Such studies require the
establishment of an equilibrium between (–)-[3H]NE
and endogenous (–)-NE and involve complex, invasive
catheterization procedures. Thus, a need has been
recognized for a method to directly examine norepinephrine turnover, as well as other processes associated
with the functional activity of the sympathetic neuron
in organs like the heart [Chiueh et al., 1983; Kopin,
1990].
One class of compounds which has been studied
for this purpose compromises simple ring fluorinated
derivatives of NE [Kirk et al., 1979]. Comparative
studies of the biological activity of simple ring
fluorinated derivatives of catecholamines relative to
the parent catecholamines have suggested that fluorine
substitution produces compounds which are promising
candidates for labeling with fluorine-18 for PET studies
of sympathetic innervation and function [Chiueh et al.,
1983; Eisenhofer et al., 1988]. For example, comparative studies of racemic 6-fluoronorepinephrine (6FNE) and NE have reported 6-FNE is a valid tracer
for the presynaptic mechanisms for norepinephrine
uptake, storage, and release. Additionally, it has been
shown that 6-fluorodopamine is taken up by sympathetic neurons and converted to (–)-6-fluoronorepinephrine by the action of dopamine-b-hydroxylase.
Even though 6-fluorodopamine is a poorer substrate
than dopamine for labeling storage pools of NE, this
observation provided the basis for recent mechanistic
PET studies with 6-[18F]fluorodopamine (6-[18F]FDA)
examining its use to image sympathetic innervation and
function in the canine heart [Goldstein et al., 1990].
The 6-[18F]FDA used in this study was of low specific
activity, however, raising issues of violation of the tracer
principle. Nevertheless, while other positron-emitting
false neurotransmitters such as [18F]fluorometaraminol
[Rosenspire et al., 1989; Schwaiger et al., 1990a;
Wieland et al., 1990], and [11C]hydroxyephedrine
235
[Schwaiger et al., 1990b, 1991] have been used to
visualize adrenergic innervation in the heart, it has
been suggested that the chemical similarity of the ring
fluorinated catecholamines to the parent catecholamines may result in tracers which label the norepinephrine storage pool and better reflect the turnover
rate of endogenous norepinephrine [Kopin, 1990].
The considerable importance of [18F]fluoride ion
for labeling radiopharmaceuticals and the biomolecules
used in PET research led us to investigate the
feasibility of using nucleophilic aromatic substitution
by [18F]fluoride ion as a more general method
applicable to aromatic substitution when both electron-donating and electron-withdrawing substituents
are present on the aromatic ring. This strategy has been
applied towards the synthesis of a variety of PET
radiopharmaceuticals, such as no-carrier-added F-18labeled DOPA and catecholamines, (–) and (+)-6[18F]fluoronorepinephrine ((–)- and (+)-6-[18F]FNE)
and 6-[18F]FDA [Ding et al., 1990]. This methodology
provided high specific activity tracers which are devoid
of the hemodynamic effects reported for low specific
activity 6-[18F]FDA [Goldstein, et al., 1990]. The
incorporation of chiral HPLC column chromatography
in the radiosynthesis of (–) and (+)-6-[18F]FNE
provided the first example of chiral separation of
radiopharmaceuticals with short half-life radionuclides
[Ding and Fowler, 1991]. Since previous studies
evaluating the behavior of 6-FNE as a tracer were
conducted with the unlabeled racemic mixture, the
availability of (–)- and (+)-6[18F]FNE has provided
the first opportunity to examine their kinetics and
metabolism.
As shown in Figure 9, there was a longer
retention of F-18 activity after intravenous administration of (–)-6-[18F]FNE to baboons, as compared to the
(+) isomer. For example, 86% of the peak F-18 heart
uptake for (–)-6-[18F]FNE was retained at 60 min
postinjection in contrast to 62% for (+)-6-[18F]FNE.
These comparative PET studies of (–)-6-[18F]FNE with
(+)-6-[18F]FNE allow an examination of the behavior
of 6-FNE within the presynaptic terminal. Previous
studies with (–)- and (+)-[14C] and [3H]norepinephrine
have shown that the neuronal uptake of NE itself is not
stereoselective [Draskoczy et al., 1968]. However, only
(–)-NE serves as a substrate for the vesicular transporter [Stjarne and von Euler, 1965]. Once it is in the
vesicle it is protected from metabolic degradation and
release from the presynaptic terminal. In contrast, (+)NE is not a substrate for the vesicular transporter and
is exposed to degradative enzymes such as monoamine
oxidase within the cytosol. For example, it has been
shown that more 3,4-dihydroxyphenylethyleneglycol
(DOPEG), less 3,4-dihydroxymandelic acid (DOMA),
236
DING AND FOWLER
have also measured the degree of recovery of FNE
uptake in the heart after both a single dose and longterm treatment with desipramine. In another PET
study we have shown that pretreatment with cocaine
profoundly inhibited NE uptake as assessed by (–)-6[18F]FNE [Fowler et al., 1994b]. Recovery was slow,
with only 48% of the baseline (–)-6-[18F]FNE uptake
being recovered at 78 min after cocaine administration,
contrasting with the very short residence time of
cocaine in the heart (t1/2 ¼ 2.5–9 min). These results
reinforce the need to understand the link between
cocaine pharmacokinetics and NE transporter function
and its relationship to cardiotoxicity.
CONCLUSIONS
18
18
Fig. 9. Uptake and clearance of F after injection of (–)-6-[ F]FNE
and (+)-6-[18F]FNE in baboon heart.
and less O-methylated metabolites were formed from
(–)-[3H]NE than from (+)-[3H]NE. The intraneuronal
distribution of the two isomers (derived from the
stereoselectivity of vesicular uptake) and the stereoselectivity of MAO as well as the enzymes which are in
series with MAO were suggested to be the reasons for
the differences between (–) and (+)-norepinephrine.
PET studies with (–)-6-[18F]FNE and (+)-618
[ F]FNE showed kinetic differences consistent with
the stereoselective behavior of norepinephrine itself.
High uptake and concentration in myocardial tissue
occurred in less than 5 min for both (+) and (–)-6[18F]FNE, consistent with the lack of stereoselectivity
of norepinephrine for uptake 1. However, a faster
wash-out of F-18 radioactivity was observed for (+)-6[18F]FNE than for (–)-6-[18F]FNE, consistent with
vesicular compartmentation of (–)-6-[18F]FNE but not
(+)-6-[18F]FNE. Additionally, more O-methylated metabolites were observed with (+)-6-[18F]FNE than with
(–)-6-[18F]FNE in our studies, paralleling the results
from previous studies using [3H]-norepinephrine [Garg
et al., 1973]. Thus, the similar initial uptake but
different clearance rates and metabolism for (–)-6[18F]FNE and (+)-6-[18F]FNE indicate the similarity
between this fluorine-substituted derivative and (–)and (+)-NE. The situation with respect to metabolite
labeling patterns and vesicular storage of 6-[18F]FDA
and related compounds is similar to that discussed in
the article by DeJesus in this issue on brain imaging of
the dopamine system.
We have also carried out comparative PET
studies of (–) and (+)-6-[18F]FNE and of 6-[18F]FDA
in the same baboon, including heart uptake, metabolism, and the effect of desipramine, a tricyclic
antidepressant drug which is an inhibitor of norepinephrine reuptake (uptake 1) [Ding et al., 1993a,b]. We
In this article we have described several PET
studies of chiral molecules which have been carried out
in our laboratory. These studies clearly show that the
pharmacokinetics of drugs rely on their chemical
structure and in some cases the enantiomers do not
follow the same path in vivo. Factors such as first-pass
effects, enzyme effects, binding to plasma protein,
transport mechanism, as well as selective binding to
receptors may play important roles in the selective
distribution of chiral drugs. These studies also illustrate
the value of PET in the assessment of regional
stereospecificity of drugs and demonstrate that PET
imaging is a suitable method to investigate the behavior
of a chiral drug in the human body and is a powerful
tool in drug development. Within this context it is
important to emphasize the essential and pivotal role
that organic synthesis has played in the progression of
the PET field over the past 20 years, during which time
PET has become an important scientific tool in the
neurosciences, cardiology, and oncology and is available in hundreds of institutions worldwide. However, it
is important to point out that PET is by no means a
mature field in terms of the study of chiral drugs. The
fact that hundreds of racemic drugs have been
developed but only a few have been labeled with
positron emitters to study their stereoselectivity
illustrates and underscores a major difficulty in radiotracer development, namely, the complexities of rapid
synthesis and chiral purification. In a sense, the issues
discussed in this review article illustrate the vital role of
nuclear medicine in clinical pharmacology. New
developments in rapid organic synthesis, enantioselective chiral synthesis, and chiral purification are needed
in order to investigate in vivo stereoselectivity of new
chiral drugs with PET. Although PET is a challenging
and expensive technology, it is exquisitely suited to
human studies, particularly to studies of the functional
and neurochemical organization of the normal human
brain and other organs, and to delineating mechanisms
HIGHLIGHTS OF BROOKHAVEN PET STUDIES
underlying neurological and psychiatric disorders and
cancers. Its use in drug research and development
holds promise in understanding the duration of action
and stereoselectivity of drugs and in facilitating drug
discovery and the introduction of new drugs into the
practice of medicine.
ACKNOWLEDGMENT
This work was carried out at Brookhaven National
Laboratory under contract DE-AC02-98CH10886 with
the U.S. Department of Energy and supported by its
Office of Biological and Environmental Research.
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