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National Institute on Drug Abuse

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

May, 1996

Research Findings

Basic Research

Common Intracellular Mechanisms in Morphine Tolerance and Thermal Hyperalgesia

Dr. Jianren Mao of Virginia Commonwealth University, in his investigation of neural and molecular mechanisms of hyperalgesia, discovered that morphine tolerance and hyperalgesia have in common certain neural substrates such as activation of N-methyl-D-aspartate (NMDA) receptor and the subsequent intracellular activation of protein kinase and nitric oxide and that these cellular and intracellular commonalities result in interactions between morphine tolerance and thermal hyperalgesia, i.e., thermal hyperalgesia develops when animals are made tolerant to the antinociceptive effects of morphine, a phenomenon that may have significant clinical implications for the treatment of painful conditions such as neuropathic pain, postoperative pain, and cancer pain.

NMDA Receptor Antagonists

NIDA grantee Dr. Charles Inturrisi and his coworkers have identified two NMDA antagonists (dextromethorphan and ketamine) with well established clinical safety and shown that both can attenuate morphine tolerance and inflammatory (formalin) pain. This "dual action" could provide an important nonopioid adjunct for use in the management of patients with combined nociceptive and neuropathic pain.

Opioid Receptor Splice Variant

NIDA grantee Dr. Charles Inturrisi and his coworkers, using RNase protection technique, PCR and Southern blot analysis have provided evidence for a mouse mu opioid receptor splice variant missing exons 2 and 3. Using a riboprobe obtained from the cloned splice variant they are comparing the distribution of this splice variant and the MOR-1 mRNA in selected regions of mouse CNS. This finding is of particular interest since it may provide an explanation for the pharmacologically defined mu receptor subtypes.

Paternal Opiate Exposure and Offspring Development

NIDA-supported researchers, Drs. Theodore Cicero, Bruce Nock and their associates from Washington University School of Medicine, St. Louis, have recently reported that acute paternal morphine exposure just before breeding with drug-naive females had no effect on fertility, but exerted negative effects on the viability and development of their offspring. These findings represent the most compelling evidence to date that paternal opiate exposure can adversely affect fetal outcome and are particularly striking as they were produced by a single injection of morphine. Cicero, T.J., Nock, B. et al., J Pharmacol Exp Therap 273: pp. 386-392, 1995.

Buprenorphine and Brain Opioid Receptor Adaptation

Recently, Dr. Carmine Coscia and his associates have found that administration of buprenorphine to rats induces a brain-regionspecific down and up regulation of opioid sites. Based on these findings, the investigators suggest that buprenorphine is a useful tool to study brain opioid receptor adaptation in vivo and the information accrued may be relevant to the mode of action of this drug in the treatment of heroin and cocaine abuse. Belcheva, M.M. et al., J Pharmacol Exp Therap, In Press, 1996.

A Novel, Rapid Method for the Determination of Anandamide Amidase Activity in Tissues

Anandamide amidase is an enzyme involved in the hydrolytic degradation of anandamide, an endogenous ligand for the brain cannabinoid receptor (CB1). Anandamide amidase is distributed in the regions of the brain where the CB1 receptors exists and can therefore serve as a marker for the presence of CB1. The amidase is also found in other organs and can be used as a marker for the presence of anandamide.

A good analytical method for the determination of the activity of amidase is currently not available. The existing methods rely on the separation and measurement of radioactive hydrolysis products. Very recently a novel, rapid, and sensitive method was developed by Dr. Markriyannis and colleagues at the University of Connecticut. The new method is based on HPLC separation and does not use radioactive substrates and extensive extraction procedures are not involved. The assay could be utilized for measuring the enzyme activity in different cells and also for determining the metabolic stability of novel anandamide analogs. This method has been further extended to the measurement of anandamide levels in different tissues. One important application for the method is that this could be adapted for high throughput screening for the discovery of anandamide amidase inhibitors. Lang et al. Analytical Biochemistry, In Press.

1-Deoxy HU-210: A Very Potent Cannabinoid with High Affinity for the CB2 Receptor

Dr. Huffman and colleagues have recently synthesized the deoxy analog (this compound lacks the phenolic -OH) of the very potent cannabinoid HU-210. The synthesis was carried out following methodology developed earlier by Dr. Huffman. The biological evaluation is carried out by Dr. Billy Martin and his colleagues. The compound exhibited unexpected potency and CB2 selectivity. The CB2 selectivity is around 30 fold. Deoxy HU-210 was also very potent in vivo in the mouse model (spontaneous activity, tail flick, and rectal temperature) and was also active in the rat drug discrimination model.

Orphanin FQ: Receptor Binding and Analog Structure Activity Relationships in Rat Brain

Dr. Houghten and colleagues have reported the preparation of a tritiated form of orphanin FQ (also known as nociceptin). This radioligand was used to develop a radioreceptor assay using rat brain homogenates. Initial kinetic studies identified a single high affinity binding site. Specific binding was found in all parts of rat brain tested. Binding was observed in the caudate nucleus, a region reported not to have expression of the mRNA of the orphanin FQ receptor.

Thirty four analogs of orphanin FQ were synthesized and tested. The loss of activity upon Nterminal truncation and the relatively unchanged affinities observed for C-terminal truncation analogs indicate that the N-terminal plays a crucial role in binding. The data supports the view of Meunier et al. that orphanin FQ binding to its receptor is analogous to dynorphin binding to the kappa receptor, with N-terminal Phe-Gly-Gly-Phe representing the "message" portion of the molecule and the C-terminal amino acids representing the "address." The binding capability of the pentapeptide analog of orphanin FQ is also similar to that seen in the opioid system, in which the pentapeptide representing the N-terminal of a longer sequence (e.g. beta-endorphin or dynorphin) is found to bind to the receptor with high affinity.

Prenatal Cocaine

Dr. Jerrold Myer of the University of Massachusetts has been studying the effects of prenatal cocaine in rats. In a series of experiments, he has determined the characteristics and localization of fetal and adult brain recognition sites labeled with [125I][RTI-55, a potent cocaine congener. His results, "Characterization and Localization of [125I]RTI-55-Labeled Cocaine Binding Sites in Fetal and Adult Rat Brain". J. Pharmac. Exp. Ther. In Press, 1996, apparently represent the first visualization of cocaine's sites of action in the fetal brain and thus provide strong evidence that cocaine can interact directly with fetal brain neurons.

Cocaine and Reward

Cocaine effects on reward mechanisms have been studied by several grantees. Drs. Friebert Weiss and Athena Markou at the Scripps Research Institute in La Jolla and Roger Spealman at the New England Regional Primate Center in Boston have been concentrating on dopaminergic mechanisms in rats and monkeys. Dr. Weiss' work with the D1, D2 and D3 agonists and antagonists has implicated the D1 binding site as particularly sensitive for cocaine "craving", while all three sites appear equally effective for cocaine "seeking" behaviors. "Effects of Dopamine Agonists and Antagonists on Cocaine-Induced Operant Responding for a CocaineAssociated Stimulus". Psychopharmacology, In Press, 1996.

Dr. Weiss has also compartmentalized cocaine self-administration in the rat to "cocaine-seeking", "operant responding", "extinction", and "reinstatement" behaviors which can be separately identified and measured. These tests may provide useful tools for the assessment of potential treatment drugs for the various parts of cocaine abuse in humans. "Measures of Cocaine-Seeking Behavior Using a Multiple Schedule of Food and Drug Self-Administration in Rats". Drug and Alcohol Dependence 38: pp. 237-246, 1995.

Dr. Markou has developed the microdialysis techniques for studying monoamine levels in the amygdala. She has reported that both dopamine and serotonin are detectable in the dialysate, although serotonin levels were not increased in response to KCl stimulation, while dopamine levels were increased. Most investigators have used the microdialysis technique exclusively in the striatum. Studies on the amygdala are particularly important because of new information that this brain structure is important in mediating drug hunger or relapse. Furthermore, the amygdala is thought to be critical in the formation of associations to the drug experience. These data will expand our overall knowledge of monoamine involvement in other brain areas. "Basal Extracellular Dopamine Levels in the Nucleus Accumbens are Decreased During Cocaine Withdrawal after Unlimited-Access Self-Administration". Originally discussed in Brain Research 593: pp. 314318, 1992.

Dr. Spealman's self-administration and discrimination work in monkeys has concentrated on the D1, D2 and D3 receptor agonists, and the development of newer D3 compounds that may have less extrapyramidal side effects than existing compounds. His work on the D1 agonists have led him to postulate that this binding site will maintain cocaine self-administration, depending on the schedule of reinforcement and the pharmacological properties (selectivity, intrinsic efficacy) of the particular drug.

"Differential Effects of D1 and D2 Receptor Agonists on Schedule-Controlled Behavior of Squirrel Monkeys. J. Pharmac. Exp. Ther. 273: pp. 40-48, 1995; "Self-Administration of D1 Receptor Agonists: Comparison Under Different Schedules of Reinforcement. NIDA Res. Monograph Series, In Press.

Other grantees have concentrated on serotonergic involvement in cocaine's rewarding effects. Dr. Nissar Darmani at the Kirksville College of Osteopathy and Medicine has been studying cocaine's effect on the rat "head-twitch" response (HTR) evoked by serotonergic drugs (5-HTP, fenfluramine, sertraline, etc.). His data suggests that 5HT-1A and 5-HT2A receptor supersensitivity results from chronic cocaine administration, resulting in marked increases in the HTR. "The Mechanism by Which the Selective 5-HT1A Receptor Antagonist S-(-)UH 301 Produces Head-Twitches in Mice. Pharmac. Biochem. Behav. In Press, 1996; "The Stimulatory and Inhibitory Components of Cocaine's Actions on the 5-HTP-Induced 5-HT2A-Receptor Response. Pharmac. Biochem. Behav. In Press, 1996.

Dr. George King of the Duke University Medical Center, has a FIRST award to study the contribution of the 5-HT3 receptor on cocaine sensitization and tolerance in rats. In the past year, he has shown that the co-administration of 5-HT3 antagonists and cocaine block the development of sensitization and tolerance. His data suggest that 5-HT3 receptor stimulation is critical for the development of sensitization and tolerance. Furthermore, his research indicates that continuous cocaine administration induces a functional down-regulation of 5-HT3 receptors in the nucleus accumbens. Since the nucleus accumbens is thought to be critical in mediating reinforcement processes in general, this down-regulation may be critical in our understanding of the consequences of cocaine abuse.

Neurotensin Modulates DA Activity

While NIDA neuroscience is focused on the dopamine (DA) systems in brain, attention is being broadened to the role in drug abuse of the systems that modulate DA function and are affected by DA release. The group headed by Glen R. Hanson, Ph.D., DDS at the University of Utah College of Pharmacy has focused on the neurotensin (NT) system. In rats, removal of extracellular NT by infusion of NT antibody into the brain, or blockade of the NT receptor by SR-48692, caused a large increase (5-fold in the case of the antibody) in the DA released in the nucleus accumbens after sc injection of a low methamphetamine dose (0.5 mg/kg), accompanied by dramatic enhancement of locomotion and rearing. These findings suggest that the endogenous NT system is activated by low doses of methamphetamine, and serves to restrain the dopamine response. In conscious, freely moving rats, in both the nucleus accumbens and striatum, NT release was increased by a DA D2 receptor agonist (quinpirole) and decreased by a D2 antagonist (eticlopride), demonstrating for the first time that D2 receptors are important in the regulation of extrapyramidal and limbic NT release. Wagstaff, Gibb & Hanson, Brain Res., 1996; The release of NT by low dose methamphetamine was blocked by both D1 and D2 antagonists in the striatum, but only by the D2 antagonist in accumbens. Wagstaff, Gibb & Hanson, J. Pharmacol. Exp. Ther., 1996.

Regulation of Opiate Receptors

NIDA MERIT awardee Dr. Robert Elde from the University of Minnesota has made great gains in defining the spatial relations between opioid peptides and their receptors. In some instances, opioid immunoreactive dendritic processes interdigitate with opioid immunoreactive terminals. In other instances (e.g. nucleus accumbens and interpeduncular nucleus) nerve terminals containing opioid peptides form networks and occur in adjacent regions to neuronal elements containing opioid receptors. These later findings suggest the possibility that opioid neurotransmission may occur over significant volumes. The use of high magnification confocal and preliminary electron microscopic studies have also suggested that a great fraction of opioid receptors are not on the plasma membrane of axons or their terminals, but rather are intra-axonal in association with vesicles, perhaps awaiting exteriorization in response to stimulation. Dado, et al. NeuroReport 5: pp. 341-344, 1993; Arvidsson, et al. J. Neuroscience 15: pp. 1215-1235, 1995; Arvidsson, et al. J. Neuroscience 14: pp. 3328-3341, 1994; Arvidsson, et al. Proc. Natl. Acad. Sci. 92: pp. 5062-5066, 1995.

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