Cocaine and the Changing Brain
Cocaine Targets In Primate Brain: Liberation From
Prosaic Views
Bertha Madras, Ph.D.
Harvard Medical School and
New England Regional Primate Center
Southborough, MA
Attributed to Hippocrates (470-377 B.C.), this
riveting quotation is a haunting description of
drug abuse and addiction:
"Men ought to know that from
the brain, and from the brain
only, arise our pleasures, joys,
laughter and jests, as well as
our pains, sorrows, griefs and
fears. It is the same thing that
makes us mad or delirious,
inspires us with dread and
fear, whether by night brings
sleeplessness, inopportune
mistakes, aimless anxieties,
absentmindedness and acts
that are contrary to habit.
These things that we are
suffer come from the brain
when it was not healthy."
Hippocrates surmised, rightfully, that the brain
was the source of pleasure and pain. What
he could not envision 2,500 years ago was
that, at the end of the 20th century, advanced
technologies would produce drugs that mimic
all the sensations that the brain produces
endogenously.
The progression of drug abuse to addiction
and recovery can be described in phases:
the acute drug phase that produces pleasure,
the addiction phase, withdrawal, and
abstinence. The first part of Hippocrates'
quotation refers to the initial state of drug
use, when sensations are positive and
incentive builds to use again. The second
part of the quote corresponds to the second,
third, and final stages of drug use-addiction,
withdrawal, and craving. This presentation
focuses on the initial phase and the initial
targets of cocaine in the brain.
Accumulating evidence indicates that
dopamine-containing neurons are principal
targets of cocaine in the brain. Dopamine is
found in neurons unevenly distributed in the
brain. At least four major clusters of cells
produce dopamine. Of these, the mesolimbic
dopamine neurons are often implicated as the
mediators of reward or reinforcement. They
originate in the ventral tegmental area and
project to various forebrain structures,
including the nucleus accumbens and cortical
regions. When dopamine is released from
these projection neurons, it activates at least
five subtypes of presynaptic and
postsynaptic dopamine receptors. Receptor
activation by dopamine is rapidly terminated
by a number of processes, of which
transport into the presynaptic neuron by the
dopamine transporter (DAT) is one of the
most significant.
After intravenous administration, cocaine
accumulates in dopamine-rich regions
(caudate-putamen and accumbens). In these
regions, a single dose of cocaine raises the
extracellular concentration of dopamine, and
the rise and decline of dopamine correspond
temporally to the cocaine levels in the blood
and brain. The increase of dopamine is
attributable to blockade of dopamine
transport, which results in an inundation of
dopamine in the extracellular fluid. At a
molecular level, the evidence is strong. The
affinities of cocaine, cocaine congeners, and
other inhibitors of the DAT for binding to the
DAT correlate highly with their potencies for
blocking dopamine transport. The relative
potencies of drugs at the DAT also
correlates, albeit not as impressively, to their
potencies for producing behavioral
stimulation, maintaining self-administration,
and engendering cocaine-like subjective
effects.
How and where on the molecule do drugs
such as cocaine bind to and block the DAT?
Can such information lead to novel drugs to
treat cocaine addiction? Is it possible to
design a drug that prevents access of
cocaine to the transporter but allows
dopamine to be transported to the interior of
the cell? Drugs targeted to the transporter
may have other uses, such as in the
treatment of Parkinson's disease and
attention deficit disorder. Transporter
research is needed to address these
fundamental questions and to provide
important leads for effective cocaine
medications. This presentation outlines two
leads, generated by this laboratory, which
compel revisions of some current
concepts-a liberation from prosaic views.
Prosaic View #1: The DAT Is A
Protein That May Form A Channel,
Structured From 12
Transmembrane Domains. Where
On This Protein Do Dopamine And
Cocaine Bind?
Two key components of the cocaine molecule
are its amine nitrogen and its aromatic ring.
Most transmitters, including dopamine,
serotonin, and norepinephrine, contain an
amine nitrogen in their structure. Exceptions
such as a newly discovered derivative of
anandamide are rare. This basic structural
element has driven our models of how
transmitters bind to receptors and
transporters and, equally importantly, has
driven drug design. The paradigm for how
dopamine binds to the DAT is borrowed from
the beta-adrenergic receptor model. If a highly
conserved aspartic acid residue on the beta-adrenergic receptor is mutated to a neutral
amino acid, the capacity of the receptor to
bind norepinephrine is lost. A model was
constructed that proposed that the amine
nitrogen of the transmitter formed an ionic
bond with the carboxylic acid residue of
aspartic acid. When a similar approach was
applied to the DAT, by mutating a highly
conserved ASP79 on the DAT, the DAT failed
to transport dopamine effectively. An
analogous model evolved for the DAT, which
proposed that dopamine (and cocaine) bind to
the DAT by the formation of an ionic bond
between the amine nitrogen of dopamine and
the aspartic acid residue. Such an ionic bond
would also serve to form the first point of
attachment between drug and transporter.
Accordingly, amine drugs must mimic actions
of the native neurotransmitter.
However, we (Peter Meltzer and author)
recently developed a new series of
compounds, based on the tropanes, that lack
an amine nitrogen. These nonamines, in
which the amine nitrogen is replaced with an
oxygen, bound the DAT with potencies similar
to those of their parent amine analogs.
Furthermore, they displayed biological activity
in a number of assays. These nonamines
also very potently inhibited DAT transport in
vitro. Nonamine represents a new class of
monoamine transporter drugs.
Structure-activity relationships indicate they
can be either highly selective for the DAT or
relatively nonselective.
What is the binding domain of these
compounds? Do they see the same acceptor
sites? Do they fit the same three-dimensional
space as their amine-bearing counterparts
such as WIN 35,428 or the GBR series,
cocaine, or mazindol? The amine-bearing
dopamine transport inhibitors display similar
pharmacological binding profiles unique to the
DAT. In this regard, the rank order of
potencies of drugs that compete for the site
on the transporter labeled by these
radioligands is similar. Is the binding profile of
[3H]O-1059, a nonamine, similar to its amine-
bearing counterpart [3H]WIN 35,428? After
radiolabeling [3H]O-1059, it was found to bind
to a single high-affinity site on the DAT. The
binding was saturable and stereoselective.
To investigate whether the three-dimensional
space occupied by [3H]O-1059 was the same
as the monoamine [3H]WIN 35,428,
competition studies with a series of potent
amine-bearing drugs were conducted. The
rank order of potency of drugs binding to the
dopamine transporter at the nonamine site
was virtually identical to the sites labeled by
[3H]WIN 35,428. It can be concluded that both
monoamines and nonamines bind to the same
architectural elements of the transporter.
Can these compelling data support the ionic
theory of ligand-transporter complex
formation? They suggest otherwise. The
premise is not valid that an amine nitrogen is
obligatory in the structure transport inhibitors;
however, it may still be necessary for the
association by dopamine.
The similar pharmacological specificity of the
two classes of compounds can still be
accounted for by hydrogen bonding between
the oxa moiety (to replace the amine nitrogen)
and the transporter in a region in close
proximity to the aspartic acid residue. To test
this hypothesis, we replaced the oxygen with
a carbon atom. This area of the molecule
cannot engage in any ionic or hydrogen
bonding. Surprisingly, the carbon-replaced
compounds bound almost as potently and
selectively as their oxa or amine
counterparts. This finding implies that the
capacity to block transport is embedded in the
three-dimensional structure of the compound
and not in the functional groups. These
results strongly suggest that an amine
nitrogen analogous to the amine nitrogen of
dopamine is not necessary for binding to or
blockade of monoamine transport. In addition,
the high dopamine selectivity of several of
these compounds supports the concept that
ionic or hydrogen bonding is irrelevant in
governing selectivity. Clearly, our current
model of drug-transporter interactions
requires modification.
Like cocaine, a representative nonamine, O-913, increased dopamine accumulation
when measured by microdialysis. It also
produced subjective effects comparable to
cocaine-like compounds in drug discrimination
studies. How do these compounds relate to
other drugs for other receptors or
transporters? There are no other comparable
drugs reported for transporters. Other
ligands exist that have activity at receptors
but bear no amine nitrogen. These include
the partial agonist and active component of
marijuana, delta9- tetrahydrocannabinol;
anandamide; the anticonvulsant valproic acid;
and the proconvulsant picrotoxin. We must
also consider that receptors can be activated
by pheromones and steroid hormones, which
bear no nitrogen in their structure. These
compounds suggest that they are the
progenitors of a new generation of
compounds targeted to transporters and
possibly receptors. It may be feasible to
design an anticocaine medication that binds to
the DAT without blocking uptake, but this
series of compounds do not fulfill this
requirement. Even if the amine nitrogen of a
drug is removed from the structure, it can still
effectively block dopamine transport.
Prosaic View #2: The Dopamine
Transporter Is The Principal
Target Of Cocaine.
Using PET imaging, Nora Volkow and Joanne
Fowler clearly demonstrated that trace doses
of cocaine bind primarily in the dopamine-rich
striatum. In our laboratory, ex vivo
autoradiography conducted with trace or high
doses of cocaine demonstrated that the
greatest accumulation of cocaine occurs in
the striatum. However, cocaine also
distributed to other targets in the brain. The
medial prefrontal cortex, hippocampus,
thalamus, and amygdala all bind cocaine even
though they contain low levels of dopamine.
PET imaging also reveals significant
accumulation of cocaine in the orbitofrontal
cortex. Are these cocaine binding sites
associated with the DAT? Are they relevant
to the behavioral effects and abuse liability of
cocaine?
To clarify the subsequent findings, we must
revisit early behavioral and binding
experiments that implicated the dopamine
transporter as a mediator of the behavioral
effects of cocaine. The potencies of drugs
for binding to the dopamine transporter are
correlated with their ability to elicit
self-administration. However, there is one
caveat to these findings. If the potencies of
DAT inhibitors that are cocaine congeners for
producing cocaine-like behavioral effects are
compared with their potencies at cocaine
binding sites, the data yield a steep binding
slope. However, noncongeners produce
shallow binding slopes, implying that the
noncocaine congeners are considerably
weaker in vivo than in vitro at the DAT.
Although pharmacokinetic considerations may
account for these observations, other
explanations may also be relevant.
We specifically examined areas poor in
dopamine transporters and assessed the
binding of cocaine congeners and
noncongeners to these regions. Cocaine and
its congeners bound to sites labeled by
[3H]cocaine in dopamine-poor areas with an
appropriate rank order of potency. However,
noncocaine congeners, dopamine,
norepinephrine, and serotonin did not bind to
these sites in DAT-poor areas. These
low-density sites were not associated with
dopamine transporters. Such sites were
found in the medial prefrontal cortex, the
hippocampus, the amygdala, and the
DAT-depleted striata of patients with
Parkinson's disease. Similarly, with PET
imaging, we found high accumulation and low
dissociation of cocaine in the orbitofrontal
cortex. Although these regions have low
affinity for dopamine itself and low affinity for
other transport inhibitors, they may contribute
to some of the psychological and behavioral
effects of cocaine, including craving and
withdrawal. These targets of cocaine may
contribute to the pharmacological effects of
cocaine, but further studies are needed to
characterize these sites. These "liberating
results" compel us to reexamine some of the
premises that have driven cocaine research
and drug design.
Acknowledgments
The author thanks collaborators Peter
Meltzer, Anna-Liisa Brownell, Susan George,
Roger Spealman, Susan Amara, Mark
Sonders, Randy Blakely, and Michael Fahey
and acknowledges the technical assistance
of Helen Panas and Keiko Akasofu and
graphics production by Sandy Talbot. The
research described herein was supported by
National Institute on Drug Abuse Research
Grant Nos. DA-06303, DA-09462, DA-11558,
DA-00304, and RR-00168.
Selected References
Canfield, D.R.; Spealman, R.D.; Kaufman, M.J.;
and
Madras,
B.K.
Autoradiographic
localization
of
cocaine
binding
sites
by
[3H]CFT
([3H]WIN 35,428) in the
monkey
brain.
Synapse
6:189-
195,
1990.
Madras, B.K., and Kaufman, M.J. Cocaine
accumulates in
dopamine-rich
regions
of
primate
brain
after i.v.
administration:
Comparison with
mazindol
distribution.
Synapse
18:261
-275,
1994.
Madras, B.K.; Pristupa, Z.B.; Niznik, H.B.;
Liang,
A.Y.;
Blundell, P.;
Gonzalez,
M.D.;
and
Meltze
r, P.C.
Nitrogen-based
drugs
are not
essential for
blockade of
monoamine
transporters.
Synapse
24:340
-348,
1996.
Meltzer, P.C.; Liang, A.Y.; Blundell, P.;
Gonzalez,
M.D.;
Chen,
Z.;
Georg
e, C.;
and
Madras,
B.K.
2-Carbomethoxy-3-aryl-8-oxabicyclo
[3.2.
1]octanes:
Potent
non-nitrogen
inhibitors of
monoamine
transporters. J
Med
Chem
40:266
1-267
3,
1997.
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