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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.


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.

