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National Institute on Drug Abuse

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

February, 1998

Research Findings

Basic Research

Studies on Sensitization to Morphine

Drugs of abuse such as cocaine, amphetamine, and morphine produce long-term changes in the nervous system that are reflected by increases in locomotor behavior and by increases in the rewarding properties of these drugs. Work by Dr. Eric Nestler and his colleagues published in Science suggest that these changes in brain function can be related in part to increases in expression of a single gene encoding an AMPA glutamate receptor subunit called GluR1. Glutamate, a neurotransmitter, is released from neurons onto dopamine neurons located in the midbrain. Glutamate activates a glutamate receptor, containing the GluR1 subunit, located on the surface of these dopamine neurons, causing the dopamine neurons to fire. The subsequent release of dopamine produces the sensation of reward and elicits locomotor activity. Repeated administration of morphine had previously been shown to be associated with increased expression of the GluR1 subunit on dopamine neurons. Since other changes in brain chemistry occur with repeated administration of morphine, it is not possible to infer from this previous work that increases in locomotor activity and the rewarding properties are due to the increases in the GluR1 receptors on dopamine neurons that might make dopamine neurons more sensitive to the excitatory effects of glutamate. To test directly the theory that the increased rewarding properties and increased locomotor activity produced by repeated morphine administration result from increased expression of GluR1 receptors, Dr. Nestler and his colleagues genetically engineered a herpes simplex virus to express the GluR1 receptors when neurons are infected with the virus. They then injected the virus in the vicinity of the dopamine neurons of rats that had not been exposed to morphine. The dopamine neurons of these naive rats became infected with the virus and expressed the GluR1 subunit. Following infection of the dopamine neurons with the genetically engineered virus, the naive rats when injected with morphine mimicked the response of rats who had been repeatedly injected with morphine. They showed increases in locomotor activity and the reinforcing effects of morphine were also increased. Rats whose dopamine neurons were injected with virus that had not been engineered or engineered to express a slightly different protein than the GluR1 receptors did not show the sensitized response to morphine. Since this experiment was done under controlled conditions, it suggests that the main mechanism by which morphine produces its enhanced responses is due to an increase in the expression of GluR1 in dopamine neurons that accompany the changes following chronic administration of morphine. These experiments might also lead scientists to test whether other abused drugs such as marijuana and alcohol increase the expression of the GluR1 subunit and increase the rewarding properties of drugs such as cocaine, morphine, and amphetamine when given repeatedly. Furthermore, the experiments by Nestler and colleagues suggest the possibility of gene therapy for addiction and other brain diseases using genetically engineered viruses. Carlezon, W.A. Jr., Boundy, V.A., Haile, C.N., Lane, S.B., Kalb, R.G., Neve, R.L., Nestler, E.J. Sensitization to Morphine Induced by Viral-Mediated Gene Transfer. Science. 277(5327), pp. 812-814, August 8, 1997.

Cocaine and fosB Mutant Mice

Chronic exposure to psychomotor stimulants such as cocaine and amphetamine produce long lasting changes in behavior that are accompanied by increases in transcription factors in a region of the brain called the striatum that includes the nucleus accumbens and the caudate putamen. These structures lie between the cortex and the midbrain. Some transcription factors activated by chronic cocaine exposure are the chronic fos related proteins. The chronic fos related proteins are derived from processing of the FosB gene. Transcription factors such as the chronic fos related proteins instruct the cellular machinery in neurons to reduce or increase the synthesis of proteins required for a particular cellular function. To test the role of the chronic fos related antigens in mediating the long lasting behavioral changes produced by cocaine, Nestler and colleagues used fosB mutant mice. Dr. Nestler in the September 16, 1997 issue of the Proceedings of the National Academy of Sciences reports that fosB mutant mice completely lack basal levels of the fos related antigens in the striatum and repeated administration of cocaine fails to produce any increase in fos related proteins. No change in striatal cytoarchitecture was observed in the mutant mice. The absence of the chronic fos related proteins was associated with increased behavioral responses to cocaine. The fosB mutant mice showed increased locomotor responses as compared to normal mice administered the same initial dose of cocaine. In addition, the mice lacking the fos related proteins showed robust conditioned place preference to a lower dose of cocaine than controls. These results suggest that the induction of the chronic fos related proteins represent a compensatory adaptation in striatal neurons that oppose the action of cocaine. Hiroi, N., Brown, J.R., Haile,.C.N., Ye, H., Greenberg, M.E., and Nestler, E.J. FosB Mutant Mice: Loss of Chronic Cocaine Induction of Fos-Related Proteins and Heightened Sensitivity to Cocaine's Psychomotor and Rewarding Effects PNAS, 94, pp. 10397-10402, 1997.

Causes of Nerve-Injury Related Pain

Dr. Allan I. Basbaum of the University of California has recently made some important discoveries reported in Science, Oct. 10, 1997 related to the causes of nerve-injury related pain. People with this "neuropathic" pain often suffer greatly, because neuropathic pain typically lasts for years and is resistant to known pain treatments. Basbaum found that in mice where the protein kinase C-gamma (PKC-g) gene was knocked out, behavioral signs of chronic pain were not present following nerve injury. However, these same mice still had normal pain responses to noxious heat stimuli. Thus, PKC-g appears to be specifically involved in the occurrence of neuropathic pain. PKC-g may be a target for new drugs to treat neuropathic pain.

Mechanism of Action of Morphine Metabolite

A recent study has suggested that there may be a different receptor for the metabolite morphine-6-glucuronide(M6G), based on non-linear ("two-site") Scatchard results on brain membrane preparations with tritiated M6G. A "biphasic" competition curve was obtained from the studies in brain tissue. Most of the binding was attributable to a lower affinity site resembling the mu receptor, with a smaller fraction attributable to a higher affinity site. The antagonist 3-methoxynaltrexone was useful in differentiating these sites, since it competes for tritiated M6G more strongly than it competes for tritiated morphine. The distribution of this potential M6G site was highest in striatum and frontal cortex. Brown, G.P., Yang, K.E., Ouerfelli, Q., Standifer, K., Byrd, D., and Pasternak, G. Journal of Pharmacology and Experimental Therapeutics, 282, pp. 1291-1297, 1997.

Biphalin Crosses Blood Brain Barrier

Biphalin, an analog of enkephalin, is a lipophilic peptide more potent than morphine as an analgesic when administered intrathecally in animals. A recent study using 125I-labeled biphalin has shown that this compound accumulates in the nucleus accumbens, pituitary, choroid plexus, cerebral spinal fluid (CSF) and lumbar region, following I.V. injection; the extent of uptake was inhibited by pretreatment with the antagonist naloxone. Secondly, the uptake of labeled biphalin was shown to be saturarable, i.e., inhibited by unlabeled biphalin, across the blood brain barrier, but non-saturable into the CSF. While the transport has not been fully explained, it is possibly based on a neutral amino acid carrier, and not on a carrier of enkephalin, or via a calcium channel. The maximum concentration of biphalin obtained in brain and CSF occurred approximately 20 minutes following injection, with 80% of the radioactivity due to intact biphalin. Abbruscato, T., Thomas, S., Hruby, V., Davis, T. Journal of Neurochemistry, 69, pp. 1236-1245, 1997.

DALDA and Obstetric Analgesia

Dr. Hazel Szeto and colleagues have recently investigated the usefulness of a peptide drug, DALDA (Tyr-D-Arg-Phe-Lys-NH2), for obstetric analgesia. DALDA is a potent and highly selective µ-agonist. In this study, Szeto et al. determined the pharmacokinetics and placental transfer of DALDA after systemic administration in sheep. DALDA was administered to pregnant and nonpregnant sheep. A one-compartment model provided an excellent fit for pregnant and nonpregnant plasma data. This data revealed that the volume of distribution of DALDA is restricted to plasma volume and that its disposition is not altered in pregnancy. DALDA was not detected in any of the plasma samples. The highly restricted placental distribution of DALDA suggests that DALDA may be a promising opioid drug for obstetrical use. Szeto, H.H., Clapp, J.F., Desiderio, D.M., Schiller, P.W., Grigoriants, O.O., Soong, Y., Wu, D., Olariu, N., Tseng, J-L and Becklin, R. In Press.

Females and Anabolic Steroid Use

Research findings, soon to be published, report that three commonly used anabolic-androgenic steroid (AAS), stanozolol, oxymetholone, or testosterone cypionate, produce alterations in the reproductive behavior and physiology of female rats. Dr. Ann Clark and her associates found AAS-induced disruptions in the cyclical display of vaginal estrus following acute or 14 days paradigm; however, their effects on sexual receptivity were different. Studies in ovariectomized rats also showed a time course variation with respect to sexual receptivity. For example, methyltestosterone, methandrostenolone and stanozolol interfered with the display of sexual receptivity on Day 14, whereas oxymetholone and testosterone cypionate had no effect. These findings illustrate that short-term administration of AAS compounds do elicit measurable and distinct effects on the estrous cycle as well as on female sexual behavior. Blasberg, M.E., Langan, C.J. & Clark, A.S. Physiology & Behavior, In Press; Clark, A.S., et al. Hormone & Behavior, In Press.

Adolescence and Anabolic-Androgenic Steroid

Dr. Richard Melloni and his group have reported recently that administration of high doses of multiple anabolic-androgenic steroids (AAS) during the adolescence period in male hamsters induced aggressive behavior as well as significant increases in arginine vasopressin (AVP) in the regions of the anterior hypothalamus (AH) implicated in the stimulation of offensive aggression. The doses of synthetic AAS selected for these experiments were similar to those heavy use self-administration regimens used by athletes. From these findings, the researchers suggest a possible role of AH-AVP in AAS-stimulated aggression. Studies are currently underway to compare the genetic and biochemical control of AVP production in the brains of AAS-exposed and control hamsters. Melloni, R.H. et al. Physiology & Behavior, 61, pp. 359-364, 1997.

Pulmonary Consequences and Marijuana/Cocaine

Recent findings published by Dr. Donald Tashkin and his group demonstrate that habitual smoking of either marijuana or cocaine significantly impairs the antibacterial and tumoricidal activities of human alveolar macrophages (AMs). In the case of marijuana, there were additional effects on the pulmonary function such as a reduction in AMs ability to produce inflammatory cytokines and a suppression of phagocytosis. While in vitro studies and animal models have long predicted some of these results, Dr. Tashkin's study provides conclusive evidence as to the immunosuppresive consequences of these drugs. These findings are important from a health perspective as the habitual smoking of these substances may enhance the susceptibility to infectious diseases, cancer, and AIDS. Baldwin, G.C., Tashkin, D.P. et al. AM J Respir Crit Care Med, 156, pp. 1606-1613, 1997.

Mice Lacking Dopamine D4 Receptors are Supersensitive to Ethanol, Cocaine and Methamphetamine

To clarify the role of Dopamine D4 receptor (D4R), NIDA Grantee David K. Grandy and his colleagues produced and analyzed mutant mice (D4R-/-) lacking this human protein. The mutant mice displayed locomotor supersensitivity to ethanol, cocaine and methamphetamine. Although less active in open field tests, the mutant mice outperformed wild-type mice on the rotorod test. Dopamine synthesis and its conversion to DOPAC were elevated in the dorsal striatum from the mutant mice. Clozapine has a high affinity for the Dopamine D4 receptor in vitro and was shown to block apomorphine-induced locomotion in rodents. Clozapine attenuated the locomotor activity of wild-type mice, but had no effect on the mutant mice. The Dopamine D4 receptor-deficient mice provide a new and useful model system to explore the in vivo role of Dopamine D4 receptor in normal and drug-induced behaviors. Rubinstein, M., Phillips, T.J., Bunzow, J.R., Falzone, T.L., Dziewczapolski, G., Zhang, G., Fang, Y., Larson, J.L., McDougall, J.A., Chester, J.A., Saez, C., Pugsley, T.A., Gershanik, O., Low, M.L., and Grandy, D.K. Cell, 90, p. 991, 1997.

Cocaine Binding Site on the Serotonin Transporter

NIDA grantee Dr. Gary Rudnick and his colleagues at Yale University have identified part of the cocaine binding site on the serotonin transporter. The Yale group replaced 20 amino acids of the transporter of the third transmembrane domain, which were deep in the interior of the protein, one at a time, with cysteine. Two of these amino acids, I172 and Y176, were close to, or in the cocaine binding site, since cocaine could protect them from reacting with a cysteine probe, MTSET. Serotonin also inhibited the labeling of both I172 and Y176 with MTSET. When MTSET was attached to either of these amino acids, it prevented the cocaine analogue (beta-CIT) from binding to the transporter. The same amino acids in the transporter involved with the binding sites for serotonin and cocaine raises the possibility that the two sites overlap considerably. The finding that serotonin and cocaine bind to the same part of the serotonin transporter suggests that the same will be true for dopamine and cocaine in the dopamine transporter and for norepinephrine and cocaine at the norepinephrine transporter. The prospect has important implications for designing drugs to combat cocaine abuse. Because cocaine itself acts by inhibiting neurotransmitter transport, any inhibitor that prevents cocaine binding may itself block transport. Chen, J.G., Sachpatzidis, A. and Rudnick, G. Journal of Biological Chemistry, 272, pp. 28321-28327, 1997.

Genetically Deficient CYP2D6 Metabolism Provides Protection Against Oral Opiate Dependence

Oral opiates (e.g. codeine, oxycodone, and hydrocodone) are metabolized by cytochrome CYP2D6 to metabolites of increased activity (e.g. morphine, oxymorphone and hydro-morphone). CYP2D6 is genetically polymorphic. Four to ten percent of Caucasians lack CYP2D6 activity (poor metabolizers) due to inheritance of two non-functional alleles. Dr. Edward Sellers of Addiction Research Foundation of Ontario, Canada, a NIDA grantee, and his colleagues tested whether the failure to activate oral opiates was a protection factor in opiate dependence by genotyping (CYP2D6*3 and CYP2D6*4 defective mutant alleles) Caucasians who met or didn't meet DSM criteria for oral opiate dependence. In opiate dependent subjects they found no poor metabolizers. In contrast, the poor metabolizer frequency in never-dependent control and multi-drug dependent comparison groups was 4% and 6.5%, respectively. This under-representation of poor metabolizers in people dependent on oral opiates suggests that the CYP2D6 defective genotype is a pharmacogenetic protection factor for oral opiate dependence. This is the first investigation and demonstration of differences in genetically determined P450 metabolism influencing risk for substance dependence. Tyndale, R.E., Droll, K.P., and Sellers, E.M. Genetically Deficient CYP2D6 Metabolism Provides Protection Against Oral Opiate Dependence. Pharmacogenetics, 7, pp. 375-379, 1997.

Pharmacological Characterization of Orphanin FQ/Nociceptin and its Fragments

The cloning of a fourth member of the opioid receptor family has led to the discovery of a new neuropeptide termed orphanin FQ or nociceptin (OFQ/N). Dr. Gavril W. Pasternak and his research team of the Memorial Sloan-Kettering Cancer Center studied OFQ/N in CD-1 mice confirming the ability of OFQ/N to rapidly induce hyperalgesia within 15 min which is insensitive to opioid antagonists. This is followed in the next 30 min by loss of hyperalgesia and the appearance of analgesia in the tailflick assay which is readily reversed by opioid antagonists. However, the very poor affinity of OFQ/N for all the traditional opioid receptors and the insensitivity of OFQ/N analgesia to antisense oligodeoxynucleotides active against MOR-1, DOR-1 or KOR-1 sequences that selectively block mu, delta or kappa1 analgesia, respectively, make it unlikely that OFQ/N analgesia is mediated through typical opioid receptors. Blockade of the antiopioid F system by haloperidol enhances the analgesic potency of OFQ/N of more than 100-fold. This effect is pronounced in BALB-C and Swiss-Webster mice. Although OFQ/N alone has little analgesic activity in these mice, the blockade of sigma systems with haloperidol uncovers a robust analgesic response in both strains. Two shorter OFQ/N fragments, OFQ/N(1-7) and OFQ/N(1-11), also are analgesic in CD-1 mice and their actions are reversed by the opioid antagonist diprenorphine despite very poor affinities of both peptides against [125I]OFQ/N binding and all the opioid receptors. In antisense studies, a probe targeting the first coding exon of KOR-3 eliminates OFQ/N hyperalgesia, but not OFQ/N analgesia. Conversely, antisense probes based on the second and third coding exons are inactive against OFQ/N hyperalgesia but readily reverse 63 opioid analgesia. These results suggest that OFQ/N elicits both analgesia and hyperalgesia through pharmacologically distinct receptors that do not correspond to traditional opioid receptors. Rossi, G.C., Leventhal, L., Bolan, E., and Pasternak, G.W. Pharmacological Characterization of Orphanin FQ/Nociceptin and its Fragments. JPET 282(2), pp. 858-865, 1997.

Chronic Morphine Exposure and the Immune System

Groups of rats were differentially pre-exposed to morphine in a drinking solution (0.2, 0.4 or 0.6 mg/ml) or to tap water 20 days prior to an acute injection of morphine (15 mg/kg) administered 1 hour prior to sacrifice. For the control group, a single acute dose of morphine resulted in significant suppression in natural killer (NK) activity, mitogen-stimulated splenic T- and B-cell proliferation, and gamma-interferon production. In the morphine pre-exposure groups, although mitogen stimulated T- and B-cell proliferation and gamma-interferon production was suppressed, NK activity was not suppressed in rats that drank the two highest concentrations of morphine. These results suggest that tolerance develops to morphine's suppressive effect on NK activity but not on other measures of immune status following a chronic regimen of morphine administration that produces tolerance to morphine's antinociceptive effects and physical dependence. West, J.P., Lysle, D.T., & Dykstra, L. Tolerance Development to Morphine-Induced Alterations of Immune Status. Drug and Alcohol Dependence, 46, pp. 147-157, 1997.

GDNF Prevents the Neurotoxic Effects of Methamphetamine on Dopamine Systems

GDNF is Glial cell line-Derived Neurotrophic Factor, a recently identified and cloned polypeptide that has striking effects on dopamine (DA) neurons. GDNF has previously shown long-lasting neurotrophic effects on DA cells in culture and on adult DA cells in vivo. In addition, Dr. Barry Hoffer, Intramural Research Program Director at NIDA, as well as several other investigators, have shown that GDNF inhibits the neurotoxicity, and promotes recovery from the neurotoxic effects, of 6-OHDA and MPTP. Wayne A. Cass, Ph.D. from the University of Kentucky College of Medicine administered GDNF unilaterally into the striatum of rats one day before they received a neurotoxic regimen of methamphetamine (METH). This METH regimen normally results in long-lasting decreases in striatal DA and serotonin function and levels in rats. It is possible that similar neurotoxic effects could occur in METH abusers. Using in vivo electrochemistry, he found that GDNF prevented the METH-induced reductions in potassium-evoked release of DA on the GDNF-treated side of the brain, a week after the METH treatment. GDNF also prevented the reduction in tissue levels of DA normally observed following this METH treatment. The ability of METH to reduce brain serotonin levels was not affected by GDNF (J. Neurosci. 16, pp. 8132-8139, 1996), emphasizing the selectivity of GDNF for DA systems. Recent studies also indicate that GDNF appears to up-regulate DA functioning in normal rats within 24 hours of administration. Soc. Neurosci. Abstr. 23, p. 56, 1997.

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