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Director's Report to the National Advisory Council on Drug Abuse
September, 2001

Research Findings

Basic Research

T-Channels on Pain Transmission Fibers Might be Novel Targets for Analgesic Agents

Although T-type calcium channels were first described in sensory neurons, their function in sensory processing has not been clearly established. The authors show that activation of these T-type calcium channels on sensory fibers induces an increased sensitivity to pain. They show that this increased sensitivity to pain is blocked by reducing the activity of these channels with 5,5'-dithio-bis-[2-nitrobenzoic acid] (DTNB). Further, DTNB by itself was able to produce analgesia to both thermal and mechanical (e.g. pinch) stimuli. These data strongly suggest that these channels are critical in the transmission of pain signals to the brain. Modulation of the activity of these channels might provide a novel method of producing analgesia, with broad clinical applications. Todorovic, S.M., Jevtovi-Todorvic, V., Meyenburg, A., Mennerick, S., Perez-Reyes, E., Ramano, C., Olney, J.W., and Zorumski, C.F. Neuron, 31, pp. 75-85, 2001.

Cocaine and Vascular Damage

Cocaine use is associated with a variety of vascular complications, including different forms of vasculitis. In a recent paper published in Life Sciences, Dr. Sulie Chang and her associates examined the actions of cocaine on the microcirculation. They observed that chronic exposure to cocaine resulted in an alteration in the hemodynamics and leukocyte-endothelial interaction in the venules of rats. They postulated that this may occur via modulation of the expression of intercellular adhesion molecule-1 mRNA. Increases in the number of total circulating white blood cells (WBCs) and WBC rolling flux changes in blood cell velocity, and an increase in inflammation-induced leukocyte-endothelial cell adhesion were also seen in animals given cocaine on a chronic basis. Thus, the hemodynamic changes elicited by cocaine may produce a decrease in the effective vessel diameter and an increase in the intravascular resistance. These effects may underlie the progressive vascular damage seen in chronic cocaine-abusing individuals. Chang, S.L., Bersig, J., Felix, B., Fiala, M., and House, S.D., Chronic Cocaine Alters Hemodynamics and Leukocyte-Endothelial Interactions in Rat Mesenteric Venules. Life Sciences, 66(24), pp. 2357-2369, 2000.

Evidence Accumulates Showing that the Mu-Opioid Receptor Plays a More Important Role in Analgesia than the Delta-Opioid Receptor

There is evidence that mu-selective opioid agonists produce less respiratory depression than delta-selective agonists. It is proposed that highly selective mu-opioid agonists may have an advantage over existing opiate analgesics such as morphine. The recent development of mu-selective opioid agonists is based on modifications of the dermorphin sequence. Degradation of dermorphin by tissue peptidases resulted in the N-terminal tetrapeptide H-Tyr-D-Ala-Phe-Gly-OH, which has a mu-selectivity similar to the parent peptide. Amino acid substitutions of this tetrapeptide led to the development of the two most selective mu-opioid agonists, DALDA and {Dmt}-DALDA. Both tetrapeptides display high binding affinity and extraordinary selectivity for the mu-opioid receptor. When administered intrathecally to rats, DALDA and Dmt-DALDA were found in the rat tail-flick test to have an analgesic potency of 14- and 3000-times respectively as compared to morphine. In addition, the duration of analgesia was significantly longer after DALDA and Dmt-DALDA administration when compared at equieffective doses. However, a potential problem with peptide analogs as therapeutic agents is their susceptibility to enzymatic degradation in vivo and short elimination half-lives. In this study, the investigators compared in sheep the stability of DAMGO, DALDA and Dmt-DALDA after systematic administration. Peptide concentrations were measured using high performance liquid-chromatography-mass spectrometry. When incubated in sheep blood at 37 degrees centigrade, all three peptides were stable over a 2-hour period. When given intravenously to sheep, the apparent volume of distribution was 80ml/kg for all three peptides, suggesting that the distribution was limited to blood volume. Plasma clearance of DAMGO was 10-fold faster than DALDA and Dmt-DALDA and their elimination half-lives were 0.24, 1.5 and 1.8 h for DAMGO, DALDA and Dmt-DALDA respectively. The half-lives of DALDA and Dmt-DALDA in sheep are even longer than morphine and meperidine. These favorable pharmcokinetic properties, together with their mu-selectivity, potency, and long duration of action, make them ideal candidates as opioid analgesics. Szeto, H.H., Lovelace, J.L., Fridland, G., Soong, Y., Fasolo, J., Wu, D., Desiderio, D.M. and Schiller, P.W. in vivo Pharmacokinetics of Selective mu-Opioid Peptide Agonists. J PET, 298(1), pp. 57-61, 2001.

Structure Activity Relationships at Monoamine Transporters and Muscarinic Receptors for N-Subsituted-3-Alpha-(3'-Chloro-, 4'-Chloro-, and 4', 4"-Dichloro-Substituted-Diphenyl) Methoxytropanes

The design, synthesis, and evaluation of 3 alpha-(diphenylmethoxy) tropane (benztropine) analogs have provided potent and selective probes for the dopamine transporter. Structure activity relationships (SARs) have been developed that contrast with those described for cocaine, despite significant structural similarity. Furthermore, behavioral evaluation of many of the benztropine analogs in animal models of cocaine abuse has suggested that these two classes of tropane-based dopamine uptake inhibitors have distinct pharmacological profiles. The benztropine analogs: in mice do not demonstrate efficacious locomotor stimulation; do not fully substitute for a cocaine discriminative stimulus; and in rhesus monkeys are not appreciably self-administered. These compounds are generally more potent than cocaine as dopamine uptake inhibitors in vitro, although their actions in vivo are not consistent with this action. These observations suggest that differing binding profiles at the serotonin and norepinephrine transporters as well as muscarinic receptors may have a significant impact on the pharmacological actions of these compounds. In addition, by varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics as well as pharmacodynamics will be directly affected. Therefore, in an attempt to systematically evaluate the impact of chemical modification on these actions, a series of N-substituted analogs of 3'-chloro-, 4'-chloro-, and 4',4"-dichloro-3-alpha-(diphenylmethoxy) tropanes were synthesized. These compounds were evaluated in rat tissue for displacement of [H-3]WIN 35,428 from the dopamine transporter, [H-3]citalopram from the serotonin transporter, [H-3]nisoxetine from the norepinephrine transporter, and [H-3]-pirenzepine from muscarininc mu receptors. SARs were developed and compared to a series of N-substituted-3 alpha-(bis-4'-flurophenyl)methoxytropanes. These present SARs followed previously reported studies with the single exception of the N-butylphenyl substituent, which did not provide high affinity binding in any if these three sets of analogs, as it did in the 4',4"-difluroseries. X-ray crystallographic analyses of these parent ligands were compared to analyses of 3 alpha-(bis-4'-flurophenyl) methoxytropane. The results provided supportive evidence that the N-substituent, in this class of compounds, is not optimal for binding at the dopamine transporter. These studies provide binding profile data that can now be used to correlate with future behavioral analyses of these compounds and may provide insight into the kind of binding profile that might be targeted as a potential treatment for cocaine abuse. Newman, A.H., Robarge, M.J., Howard, I.M., Wjttkopp, S.L., George, C., Kopajtic, T., Izenwasser, S, and Katz, J.L.. J. Med. Chem. 44 (4), pp. 633-640, 2001.

Prenatal Exposure to Methamphetamine Increases the Male Offspring's Vulnerability, When Adults, to Methamphetamine's Neurotoxicity, in Mice

The use of club drugs, such as methamphetamine, by women of childbearing age has become a public health concern. Dr. Alfred Heller and his colleagues at the University of Chicago and Illinois Institute of Technology, modeled this situation in mice. They administered a neurotoxic dose of methamphetamine to pregnant mice during gestational days 7 to 18. Fetal exposure alone did not produce neurotoxicity. The researchers also gave methamphetamine to the offspring after they had become young adults (11 weeks of age), at minimally neurotoxic doses. They observed an enhanced neurotoxicity in the male offspring when they were injected with methamphetamine as adults. The neurotoxicity was evidenced by greater methamphetamine-induced lasting reductions of dopamine and its metabolites in the striatum and of dopamine in the ventral brainstem. Some effects of prenatal methamphetamine exposure were observed in female offspring, but these were much less than those seen in males. The ability of methamphetamine to induce neurotoxicity is associated, in part, with its ability to raise body temperature (hyperthermia). These doses of methamphetamine did not raise the body temperature of the adult female offspring, while the methamphetamine injections did raise the body temperature of the adult males. However, the hyperthermic response to methamphetamine was the same in the adult males whether or not they had been exposed to methamphetamine in utero. These findings raise the concern that male methamphetamine abusers may have an enhanced risk for the neurotoxic effects of the drug if they were previously exposed to it in utero. Furthermore, the mother's methamphetamine abuse may predispose her male offspring to other neuropathological disorders, such as Parkinson's disease. Heller, A., Bubula, N., Lew, R., Heller, B., and Won, L. Gender-Dependent Enhanced Adult Neurotoxic Response to Methamphetamine Following Fetal Exposure to the Drug. J. Pharmacol. Exp. Ther., 298 (2), pp. 1-11, 2001.

Activation of Nicotine Receptors May Contribute to Cocaine Reinforcement

Dr. Marina Picciotto and her colleagues at Yale University examined the possible role of nicotinic cholinergic receptors (nAChR) in cocaine place preference, a measure of reinforcement, in mice. Nicotine and the psychomotor stimulants are thought to exert their reinforcing properties via an action on the mesolimbic dopamine system. When animals were given both a low dose of cocaine and the nicotinic antagonist mecamylamine, place preference was disrupted. In mice lacking the high affinity nAChR containing the β2 subunit, the researchers also found a diminished place preference to cocaine. By contrast, when a low dose of nicotine was administered, place preference was potentiated to a previously subthreshold dose of cocaine. Measures of dopamine levels and metabolism were decreased in normal mice following cocaine treatment, but mice lacking the β2 subunit showed no such effect. Zachariou, V., Caldarone, B.J., Weathers-Lowin, A., George, T.P., Elsworth, J.D., Roth, R.H., Changeux, J.P., and Picciotto, M.R. Nicotine Receptor Inactivation Decreases Sensitivity to Cocaine. Neuropsychopharmacology, 24(5), pp. 576-89, 2001.

Transport of Opioids from the Brain to the Periphery by P-Glycoprotein: Peripheral Actions of Central Drugs

Many peptides and transmitters found within the brain also have peripheral sites of action. Dr. Pasternak and his research team now demonstrate that the brain releases functionally active neurotransmitters/neuromodulators directly into the blood through a saturable P-glycoprotein (Pgp) transport system. Downregulating Pgp1 expression with antisense reduced the brain-to-blood transport of morphine, β-endorphin and other opioids. Lowering Pgp expression significantly enhanced systemic morphine analgesia and prevented tolerance, but diminished the analgesic activity of centrally administered morphine. This implies that supraspinal analgesia resulted from a combination of central and peripheral mechanisms activated by morphine transported from the brain to the blood. Similarly, mice with a disruption of the multiple drug resistance (Mdr1a) gene were more sensitive to systemic morphine and less sensitive to morphine given centrally. This ability of the Pgp transport system to pump functionally active compounds from the brain to the periphery defines a potentially important mechanism for modulating peripheral systems via the central nervous system. King, M., Su, W., Chang, A., Zukerman, A. and Pasternak, G.W. Nature Neuroscience, 4(3), pp. 268-274, 2001.

Chronic Morphine Induces Concomitant Phosphorylation and Altered Association of Multiple Signaling Proteins: A Novel Mechanism for Modulating Cell Signaling

Traditional mechanisms thought to underlie opioid tolerance include receptor phosphorylation/down-regulation, G-protein uncoupling, and adenylyl cyclase superactivation. A parallel line of investigation indicates that opioid tolerance development results from a switch from predominantly opioid receptor G(i alpha) inhibitory to G(beta gamma) stimulatory signaling. This results, in part, from the increased relative abundance of G(beta gamma)-stimulated adenylyl cyclase isoforms as well as from a profound increase in their phosphorylation. Dr. Gintzler and his group have demonstrated that chronic morphine administration results in the concomitant phosphorylation of three key signaling proteins: G protein receptor kinase (GRK) 2/3; beta-arrestin; and G(beta). These results were obtained using guinea pig longitudinal muscle myenteric plexus tissue. Augmented phosphorylation of all three proteins is evident in immunoprecipitate obtained by using either anti-GRK2/3 or G(beta) antibodies. However, the phosphorylation increment is greater in immunoprecipitate obtained with G(beta) antibodies. Analyses of co-immunoprecipitated proteins indicate that phosphorylation of GRK2/3, beta-arrestin, and G(beta) has varying consequences on their ability to associate. As a result, increased availability of and signaling via G(beta gamma) could occur without compromising the membrane content, and presumably the activity, of GRK2/3. Induction of the concomitant phosphorylation of multiple proteins in a multimolecular complex with attendant modulation of their association represents a novel mechanism for increasing G(beta gamma) signaling and opioid tolerance formation. Chakrabarti, S., Oppermann, M., and Gintzler, A.R. Proc. Natl. Acad. Sci. USA, 98(7), pp. 4209-4214, 2001.

Ascorbic Acid Prevents 3,4-Methylenedioxymethamphetamine (MDMA)-Induced Hydroxyl Radical Formation and the Behavioral and Neurochemical Consequences of the Depletion of Brain 5-HT

MDMA-induced 5-HT neurotoxicity has been proposed to involve oxidative stress due to increased formation of hydroxyl radicals. Recently, MDMA-induced 5-HT neurotoxicity was shown to be accompanied by a suppression of behavioral and neurochemical responses to a subsequent injection of MDMA. In the present study, Dr. G.A. Gudelsky and his colleagues examined whether suppression of the MDMA-induced formation of hydroxyl radicals by the antioxidant ascorbic acid, attenuates both the MDMA-induced depletion of 5-HT and the functional consequences associated with this depletion. They found that treatment of rats with ascorbic acid suppressed the generation of hydroxyl radicals, as evidenced by the production of 2,3-dihydroxybenzoic acid from salicylic acid, in the striatum during the administration of a neurotoxic regimen of MDMA. Ascorbic acid also attenuated the MDMA-induced depletion of striatal 5-HT content. In rats treated with a neurotoxic regimen of MDMA, the ability of a subsequent injection of MDMA to increase the extracellular concentration of 5-HT in the striatum, elicit the 5-HT behavioral syndrome, and produce hyperthermia was markedly reduced compared to the responses in control rats. The concomitant administration of ascorbic acid with the neurotoxic regimen of MDMA also prevented the diminished neurochemical and behavioral responses to a subsequent injection of MDMA. Finally, a neurotoxic regimen of MDMA produced significant reductions in the concentrations of vitamin E and ascorbic acid in the striatum and hippocampus. Thus, the MDMA-induced depletion of brain 5-HT and the functional consequences thereof appear to involve the induction of oxidative stress resulting from an increased generation of free radicals and a diminished antioxidant capacity in the brain. Shankaran, M., Yamamoto, B.K., and Gudelsky, G.A. Synapse, 40(1), pp. 55-64, 2001.

Modulation of Immune Function by Opioid Systems

Three types of opioid receptors have been characterized, primarily in neural cells. Both the kappa and delta receptors have been studied and are well characterized as reported in the following papers. Most previous studies have utilized macrophages, microglia or complex culture systems to study these effects. Papers reported herein now demonstrate that T-cells alone can modify HIV infection. The first describes more relevant functionality of the delta system in human immune T cells. The paper describes the ability of the delta opioid system to inhibit growth of HIV in thymocyte derived lymphocytes in culture. This study and others demonstrate a clear role for these systems in immune modulation. The delta opioid receptors (DORs) modulate T cell proliferation, IL-2 production, chemotaxis, and intracellular signaling. Moreover, in DOR-transfected Jurkat cells, delta opioids have been shown to suppress HIV-1 p24 antigen (Ag) expression. These observations led investigators to characterize the expression of DORs by human peripheral blood T cells and to determine whether a specific DOR agonist, SNC-80, can suppress p24 Ag expression by HIV-1-infected CD4+ T cells obtained from normal donors. By immunofluorescence flow cytometry, PHA stimulated the expression of DOR in the peripheral blood mononuclear cells (PBMC) population by 48. DOR expression was approximately 40% of both the PHA-stimulated CD4+ and CD8+ T cell subsets, and virtually all DORs were found on these subsets. To determine whether activated DORs suppress HIV-1 expression, PBMC were prestimulated with PHA, and then CD4+ T cells were purified, pretreated with SNC-80, and infected with HIV-1. In a concentration-dependent manner, SNC-80 inhibited production of p24 Ag. SNC-80 10(-10) M maximally suppressed both lymphocytotropic (HIV-1) and monocytotropic (SF162) strains; higher concentrations were less effective. Naltrindole, a selective DOR antagonist, abolished the inhibitory effects of SNC-80. Kinetic studies indicated that 24-h pre- or postincubation with SNC-80, relative to infection with HIV-1, eliminated its suppressive effects. Thus, stimulating the DORs expressed by activated CD4+ T cells significantly suppressed the expression of HIV-1. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive to standard antiviral approaches to HIV-1 infection. Sharp, B.M., McAllen, K., Gekker, G., Shahabi, N.A., and Peterson, P.K. Immunofluorescence Detection of Delta Opioid Receptors (DOR) on Human Peripheral Blood CD4+ T Cells and DOR-dependent Suppression of HIV-1 Expression. J Immunol, 167(2), pp. 1097-1102, July 15, 2001.

In the second paper, the kappa opioid system is shown to inhibit the growth of HIV in the same human T cell culture system. Thus, both opioid systems may be utilized clinically to combat HIV and other diseases. Further studies are needed to clarify this potential role. Synthetic K-opioid receptor (KOR) agonists have been shown to suppress HIV-1 expression in acutely infected macrophages. In the present study, investigators examined the effects of the KOR ligand (U50,488) on HIV-1 expression in CD4(+) lymphocytes, the main target cell of this virus. When U50,488 was added to activated CD4(+) lymphocytes, HIV-1 expression was inhibited in a concentration- and time-dependent manner with maximal suppression (60%) at 10(-7) M U50,488. The KOR selective antagonist nor-binaltorphimine (nor-BNI) had no effect by itself on viral expression but blocked the antiviral property of U50,488, suggesting that U50,488 was acting via a KOR-related mechanism. Support for the involvement of KOR was provided by the findings that 34% of activated CD4(+) lymphocytes were positive for KOR, using an immunofluorescence technique, and that seven additional synthetic KOR ligands also inhibited HIV-1 expression. The results of this study broaden our understanding of the antiviral properties of KOR ligands to include cells outside of the nervous system and suggest a potential role for these agents in the treatment of HIV-I infection. Peterson, P.K., Gekker, G., Lokensgard, J.R., Bidlack, J.M., Chang, A.C., Fang, X.G., and Portoghese, P.S. Kappa-opioid Receptor Agonist Suppression of HIV-1 Expression in CD4(+) Lymphocytes. Biochem Pharmacology, 61, pp. 1145-1151, 2001.

The third paper describes the role of the mu opioid system. It has not yet been demonstrated to directly inhibit HIV replication. However, it has been demonstrated to inhibit chemokine function, a key system involved in HIV entry into the cell. The mu opioid system seems to be a major system involved in chemotaxis inhibition. This paper reports a strong action by maturing T-cells. The maturation period of T-cells may be the time when most opioid systems maximally affect immune function. Authors have examined the chemotactic responsiveness of thymocytes to selective mu-, kappa-, and delta -opioid agonists. Results show that developing T cells migrate in response to mu- but not kappa- or delta -opioids. The mu -opioid response appears to be dependent on the classical mu -opioid receptor (MOR-1) since the chemotactic response is blocked by a selective mu-opioid antagonist, and is absent in thymocytes from MOR-l-deficient mice. Flow cytometric analysis of the mu-opioid responsive cells shows that these cells consist predominantly of highly immature CD4(-)CD8(-) T cells. These results represent the first demonstration of the functional expression of mu-opioid receptors by developing T cells. McCarthy, L., Szabo, I., Nitsche, J.F., Pintar, J.E., and Rogers, T.J. Expression of Functional Mu-opioid Receptors during T Cell Development. J Neuroimmunology, 114, pp. 173-180, 2001.

Modulation of Immune Function by Cannabinoid Systems

A more basic understanding of drug actions comes from studies of second messenger systems coupled to the cannabinoid receptors; which may mediate their action. A recent paper demonstrates some involvement of the camp signalling system but these receptors are also linked with map kinases involved in Ca transport in modulating IL-2 activity. Thus, these studies show another receptor-coupled system present in immune cells to elicit their action. Cannabinoid compounds inhibit the cAMP signalling cascade in leukocytes. One of these compounds, cannabinol (CBN has been shown to inhibit interleukin-2 (IL-2) expression and the activation of cAMP response element binding protein (CREB) and nuclear factor for an immunoglobulin chain in B cells (NF-kappaB) following phorbol-12-myristate-13 acetate (PMA) plus ionomycin (Io) treatment of thymocytes. Therefore, the objective of the present studies was to determine the role of cAMP and protein kinase A (PKA) in the CBN-mediated inhibition of IL-2, CREB, and NF-kappaB in PMA/Io-activated thymocytes. The inhibition of CREB/ATF-1 phosphorylation, or cAMP response element (CRE) or KB DNA binding activity produced by CBN in PMA/Io-activated thymocytes, could not be reversed by DBcAMP (brominated camp) costimulation. Furthermore, DBcAMP failed to reverse the concentration-dependent inhibition of IL-2 protein secretion by CBN. Pretreatment of thymocytes with compound H89 produced a modest inhibition of PMA/Io-induced CREB/ATF-1 phosphorylation and CRE DNA binding activity but H89 had no effect on protein binding to a kappaB motif. Additionally, H89 modestly inhibited PMA/Io-induced IL-2 secretion. In light of the modest involvement of the cAMP pathway in CBN-mediated inhibition of CREB and IL-2 in PMA/Io-activated thymocytes, PD098059 (PD), the MEK inhibitor, was utilized to determine the role of ERK MAP kinases in thymocytes. ERKs play a critical role in IL-2 production but not for CREB phosphorylation. Collectively these findings suggest that CBN may modulate several signalling pathways in activated T cells. Herring, A.C., Kaplan, B.L.F., and Kaminski, N.E. Modulation of CREB and NF-kappa B Signal Transduction by Cannabinol in Activated Thymocytes. J Cellular Signalling, 13, pp. 241-250, 2001.

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