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National Institute on Drug Abuse

Director's Report to the National Advisory Council on Drug Abuse

February 1997

Research Findings

Basic Research

Brain Substrates of Cocaine Addiction
Dr. Nora Volkow and her colleagues at the Brookhaven National Laboratory examined the rate of clearing of cocaine and methylphenidate in the brains of baboons. They found a dramatic difference between the two drugs, with cocaine being cleared much more rapidly than methylphenidate. Since cocaine tends to induce binging behavior whereas methylphenidate does not, Volkow and her colleagues postulate that (a) it may be the rapid uptake of these two (dopamine transporter inhibiting) drugs that gives rise to the initial pleasurable experience, and that (b) the extremely rapid clearing of cocaine sets the stage for the binging behavior exhibited by addicts. Drs. Volkow and colleagues also identified novel longer-term changes induced by cocaine withdrawal in addicts. A transient increase in the metabolic activity within the orbitofrontal cortex, in the thalamus, and in the striatum occurs within 2­p;4 weeks following detoxification, whereas a longer-term (1­p;4 month) depression in metabolic activity is seen in these identical brain regions. The authors propose that the early secondary activation (effects seen within 2­p;4 weeks) seen in drug addicts might account for the dramatic difference in brain functioning known to exist between the occasional drug user and the cocaine addict. Volkow, N.D., Ding, Y.-S., Fowler, J. S., & Wang, G.-J. Cocaine Addiction: Hypothesis Derived from Imaging Studies with PET. J. Addictive Diseases, 1996.

Neuronal Plasticity Following Chronic Cocaine
A recent paper by Dr. Ann Graybiel and her colleagues at MIT (Neuron 17: pp. 147-156, 1996) showed that repeated cocaine administration to rats is accompanied by network-level changes in the expression of the Fos-Jan family of proteins in the basal ganglia that occur with the known time course of behavioral sensitization to the drug. These findings suggest an enduring functional reorganization of circuits in the basal ganglia following extended cocaine administration that might underlie the stereotyped behaviors typical of a sensitized animal. These same basal ganglia circuits are known to connect to cortical regions that have been implicated in humans with an altered metabolic activity associated with abnormal repetitive behavioral patterns.

Distribution of Mu and Kappa Opioid Receptors
NIDA grantees Dr. Howard Fields and Dr. Zhizhong Pan (University of California, San Francisco) have recently made significant gains in both opioid pharmacology and pain research. Dr. Fields, a pioneer in pain research, first described midbrain "on" cells (that turn on analgesia) and "off" cells (that turn off analgesia). Understanding how these cell types work is essential in our understanding of how the nervous system controls pain. At the 1996 Society for Neurosciences meeting, Drs. Fields and Pan presented data showing that mu, but not kappa, opioid receptors are located upon "on" cells; whereas kappa, but not mu, opioid receptors are located upon "off" cells. These differences help to explain why mu and kappa opioid-receptor activation have different effects on pain even though both mu- and kappa-opioid receptors are coupled to G proteins and act by modifying potassium channels. Understanding the functional neural basis of the analgesic effects of opioids will allow researchers to better target these neural systems through pharmacological means.

Drugs of Abuse and Neuronal Effects
Prenatal cocaine exposure has the potential to produce abnormal development of the nervous system and cause behavioral dysfunction. Levitt and colleagues (Jones et al., Cerebral Cortex, 6: pp. 431-445, 1996) examined the effects of cocaine on prenatal development by administering intravenous cocaine to pregnant rabbits. Their analysis of neural development centered on two regions of the cerebral cortex, the anterior cingulate and primary visual cortex, in which dopamine afferents, a target of cocaine, are differentially distributed. All postnatal rabbits exposed to cocaine prenatally displayed normal features of cortical organization such as lamination patterns, cytoarchitectonic differentiation, and cortical thickness. However, the structure and organization of dendrites in the cingulate cortex but not the primary visual cortex were altered. Less than fifty percent of the apical dendrites in cocaine-exposed animals extended into the layers II and III of the cortex as compared to control animals. The dendrites of exposed animals course through the cortex in an irregular and wavy manner instead of being strait and bundled. These data suggest that exposure to cocaine in utero can produce long lasting morphological changes affecting specific brain regions. The study also suggests that structural changes following prenatal cocaine exposure may also be found in the cingulate cortex of humans.

Chronic Morphine Induces Visible Changes in the Morphology of Mesolimbic Dopamine Neurons
Mesolimbic dopamine neurons arising from the ventral tegmental area (VTA) of the midbrain play an important role in opiate mediated reward and addiction. Previous work has shown that chronic exposure to opiates produce biochemical adaptations in this brain region. In a recent paper (Sklair-Tavron, et al., Proceedings of the National Academy of Sciences, 93: pp. 11202-11207, 1996), Dr. Eric Nestler and colleagues report that these biochemical changes are associated with morphological changes in VTA dopamine neurons. Chronic morphine treatment resulted in 25% reduction on average in the area and perimeter of VTA neurons. The opioid antagonist, naltrexone, or infusion of brain-derived growth factor prevented the structural changes from occurring in VTA dopamine neurons. In contrast, chronic morphine did not affect the size of non-dopaminergic neurons or change the number of dopamine neurons in the VTA. These data suggest structural changes in dopamine neurons produced by chronic morphine exposure may be related to the specific behavioral features of addiction, and lead to new approaches for the treatment of addictive disorders.

Anabolic Steroids & Sexual Behavior
NIDA supported research reports that different anabolic-androgen steroid (AAS) compounds have quite distinct effects on male sexual behavior. For example, administration of the 17 alpha-methylated compounds had the most deleterious effects in intact male rats; while, the 19-nortestosterone esters had minimal effects on sexual behavior at any dose. The individual AAS effects in male rat copulatory behavior were closely related to their effects on serum testosterone levels. In castrated male rats, in general the alpha-methylated compounds were relatively ineffective in maintaining sexual behavior. These findings suggest that examination of individual compounds is necessary as it provides a strong foundation for analysis of the physiological and behavioral responses to AAS combinations typically administered by human users. Clark, A.S., Harrold, E.V. & Fast, A.S., Hormone & Behavior, In Press; Clark, A.S. & Fast, A.S., Behavioral Neuroscience, 110, pp. 1-9, 1996.

Other recent findings in female rats also demonstrate disruption of estrous cyclicity and reproductive behavior when different AAS compounds are administered for a short-term at levels commonly used by humans. Blasberg, M.E. Langan, C.J. & Clark, A.S., Physiology & Behavior, In Press.

Mediation of Supersensitization of Adenylyl Cyclase via G
Dr. Zvi Vogel, a NIDA grantee, has reconstituted the ability of opioids to modulate adenylyl cyclase (AC) activity in COS-7 cells. COS-7 cells cotransfected with AC type V (AC-V) and -opioid receptor cDNAs display acute opioid inhibition of AC-V activity. Prolonged exposure to -opioid receptor agonists leads to a time-dependent development of supersensitization of AC-V, which is gradually lost following withdrawal of the agonist. The supersensitization can be prevented by pertussis toxin pretreatment, indicating the involvement of Gi/o proteins, or by co-transfection with scavengers of G dimers, indicating a role for G in AC supersensitization. Contrary to several other G-dependent signal transduction mechanisms (e.g. MAP kinase), AC-V supersensitization is not affected by the Ras dominant negative mutant N17-Ras. AC-V is localized in brain areas which are involved in reward pathways and drug addiction.

Elucidation of the precise mechanism of AC supersensitization should pave the way to a better understanding of and the design of strategies for preventing opioid abuse. Avidor-Reiss, T., ...and Vogel, Z., J. Biol. Chem., 271: pp. 21309-21315, 1996.

Interactions Between Ifenprodil and the NMDA Receptor(NR)2B Subunit of the N-methyl-D-Aspartate (NMDA) Receptor
Dr. Michael Gallagher and colleagues, using chimeric NR2A/NR2B subunits co-expressed with NRlA, have localized a determinant for high affinity ifenprodil interaction to a single amino acid (Arg-337). Ifenprodil had also previously been thought to be an antagonist at the polyamine site. Experiments with chimeric and mutant receptors have shown that Arg-337 is independent of NR2B-specific polyamine stimulation. Furthermore, it was shown that polyamine stimulation depends on the expression of NR1 splice variants, whereas high affinity ifenprodil inhibition is independent of NR1 isoform expression. These studies provide evidence that ifenprodil and polyamines interact at discrete sites on the NR2B subunit. Gallagher, M.J. Huang, H., Pritchett, D.B., and Lynch, D.R., J. Biol. Chem. 271: pp. 9603-9611, 1996.

The mapping of the binding of distinguished determinants for ifenprodil within the N-terminus of the NR2B subunit is significant in that (1) it helps to understand how the amino terminal segment of the NR2B subunit interacts with the ion channel in an allosteric fashion and (2) it also has direct applications to the development of new pharmacological agents at NMDA receptors.

Novel Receptor Mechanisms for Heroin and Morphine-6-Glucuronide Analgesia
NIDA grantee Dr. Gavril W. Pasternak of Sloan-Kettering Memorial Cancer Center and Cornell University Medical College and his research team have obtained evidence, using a number of different paradigms, indicating that heroin actions are most likely not mediated through the same receptors as morphine. This finding is echoed by Dr. James M. Fujimoto's observations (another NIDA grantee) that morphine, 6-monoacetylmorphine (6-MAM) (both are active metabolites of heroin), and heroin are found to exhibit unique receptor selectivities at both spinal and supra-spinal levels as well as in different mouse strains.

The rapid metabolism of heroin to 6-MAM and its slower conversion to morphine has led many to believe that heroin and morphine act through the same receptors and that the differences between them are due to their pharmacokinetics. Dr. Pasternak and his team now present evidence strongly implying that heroin and two potent drugs, fentanyl and etonitazine, act through a unique receptor mechanism similar to morphine-6-glucuronide which is readily distinguished from morphine. Heroin, 6-MAM and morphine-6-glucuronide show no cross tolerance to morphine in a daily administration paradigm, implying distinct receptors. Strain differences also reveal differences among the drugs. CXBK mice, which are insensitive to morphine, retain their sensitivity to heroin, 6-MAM, morphine-6-glucuronide, fentanyl and etonitazine. Antisense mapping of the opioid receptor MOR-1 reveals that oligodeoxynucleotide probes against exon 2, which are inactive against morphine analgesia, block morphine-6-glucuronide, heroin, fentanyl and etonitazine analgesia. Finally, an antisense probe targeting Gi1 blocks both heroin and morphine-6-glucuronide, but not morphine analgesia. These results indicate that heroin, 6-MAM, fentanyl and etonitazine all can produce analgesia through a novel analgesic system which is similar to that activated by morphine-6-glucuronide. Rossi, G.C., Brown, G.P., Leventhal, L., Yang, K. and Pasternak, G.W. Neuroscience Letter, 216: pp.1-4, 1996.

Catalytic Antibodies
In a continuing effort to develop catalytic antibodies for the hydrolysis of cocaine, researchers at the Seattle Biomedical Research Institute have prepared synthetically a new cocaine transition state analog, which potentially may be metabolically more stable than previous analogs, particularly at the C2 position, and which will exhibit a useful immunogenic "lifetime", after linking the analog to an immunogenic protein. Berkman, C. E., Underiner, G. E., and Cashman, J. R. Journal of Organic Chemistry, 61, pp. 5686-5689, 1996.

Methadone and Hair Analysis
An analytical method has been described for the determination of methadone, and its two major metabolites, utilizing a gas chromatography/ mass spectrometry procedure, and validated in human and animal hair samples. The method may prove usable in detection and pharmacokinetic studies. Wilkins, D.G., Nagasawa, P.R., Gygi, S. P., Foltz, R. L., and Rollins, D. Journal of Analytical Toxicology, 20, pp. 355-361, 1996.

Dopamine Transporter Ligands
A series of substituted diphenylmethoxyethyl piperidines have been synthesized, and shown to be effective as potent ligands in terms of binding to the dopamine transporter, as compared to the known inhibitor GBR 12909. They also demonstrate selectivity for the dopamine as compared to the serotonin transporter. Such compounds have a potential in treating cocaine addiction. Dutta, A., Coffey, L. L., and Reith, M. E. Journal of Medicinal Chemistry, In Press.

Peptide Delivery to the Brain
A recent report describes the delivery of the pentapeptide DADLE to the brain, based on reacting the N-terminal amino acid with a nicotinolyl proline, and converting the C-terminus to a lipophilic ester for transport across the blood brain barrier. The nicotinoyl group is metabolically oxidized after transport to a lipid insoluble salt, which serves to "lock" the peptide in the brain, and the proline serves as a site designed for peptidase cleavage, releasing the esterified DADLE, and finally DADLE itself after lipase cleavage.

The DADLE delivered in this fashion showed analgesic activity in animals, which was reversed by administration of naloxone. Bodor, N., Prokai, L., Prokai-Tatrai, K. Journal of Medicinal Chemistry, 39, pp. 4775-4782, 1996.

Dopamine Dl, D2, Beta2-Adrenergic and Serotonin 5-HT1B Receptors Exist as Dimers
NIDA grantee Philip Seeman and his coworkers at the University of Toronto, Canada obtained direct physicochemical evidence that dopamine D2 and Dl, beta2 adrenergic and serotonin 5-HT1B receptors exist as dimers: Immunoblots of crude membranes from human caudate nucleus revealed that the dopamine D2 dimer exists as the predominant species. The G-protein coupled receptor (GPCR) dimers were stable in SDS and under reducing conditions indicating that dimerization was not attributed to covalent disulfide bonds. Incubation of the GPCR dimers with receptor-specific peptides derived from the putative transmembrane domains, or incubation under high temperatures or low pH resulted in the dissociation of dimers to monomers. Dopamine D2 transmembrane peptides were unable to dissociate dopamine Dl receptor dimers or serotonin 5-HT1B receptor dimers, suggesting that receptor dimers are formed by specific intermolecular noncovalent interactions involving transmembrane regions. Exposure of living D2 expressing cells (D2/cells) to glutaraldehyde resulted in a conversion of receptor monomers to dimers due to the irreversible cross-linking of receptor monomers. D2 dimers and monomers were labeled by benzamide antagonists whereas only monomers were labeled by butyrophenone antagonists, which explains the discrepancy in receptor densities estimated by these ligands in PET studies. Furthermore, dopamine exposure of D2/cells mediated an increase in cell surface D2 monomers and dimers involving the translocation of intracellular receptors. These results indicate that D2 and other G protein-coupled receptors exist as receptor dimers and that dimers play a functional role in the response to agonist exposure. These findings have important implications for neurological diseases since it is known that demolition of dimerization of the androgen receptor through mutation is one of the causes of Reifenstein syndrome in humans. Biophys. Biochem. Res. Comm., Vol 227, pp. 200-204, 1996.Dr. Michael Gallagher and colleagues, using chimeric NR2A/NR2B subunits co-expressed with NRlA, have localized a determinant for high affinity ifenprodil interaction to a single amino acid (Arg-337). Ifenprodil had also previously been thought to be an antagonist at the polyamine site. Experiments with chimeric and mutant receptors have shown that Arg-337 is independent of NR2B-specific polyamine stimulation. Furthermore, it was shown that polyamine stimulation depends on the expression of NR1 splice variants, whereas high affinity ifenprodil inhibition is independent of NR1 isoform expression. These studies provide evidence that ifenprodil and polyamines interact at discrete sites on the NR2B subunit. Gallagher, M.J. Huang, H., Pritchett, D.B., and Lynch, D.R., J. Biol. Chem. 271: pp. 9603-9611, 1996.

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