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

Research Findings

Intramural Research

Chemistry & Drug Metabolism Section, Clinical Pharmacology & Therapeutics Research Branch

Monitoring Opiate Use in Substance Abuse Treatment Patients by Sweat and Urine Testing

Although urine testing remains the standard for drug use monitoring, sweat testing for drugs of abuse is increasing. This study was designed to compare the efficacy of sweat testing versus urine testing for detecting drug use. Paired sweat patches applied and removed weekly were compared to consecutive urine specimens collected three times per week from patients in a methadone maintenance outpatient treatment program. Patches were analyzed by ELISA immunoassay for opiates and by GC/MS. Urine specimens were subjected to qualitative analysis by EMIT. Opiates were detected in 38.5% of the sweat patches with the ELISA screen. GC-MS analysis confirmed 83.4% of the screen-positive sweat patches for heroin, 6-acetylmorphine, morphine and/or codeine and 90.2% of the screen-negative patches. Heroin and/or 6-acetylmorphine were detected in 78.1% of the GC/MS positive sweat patches. Agreement in paired sweat patch test results was 90.6% by ELISA analysis. Analysis of sweat patches provides an alternate method for objectively monitoring drug use and provides an advantage over urine drug testing by extending drug detection times to one week or longer. In addition, identification of heroin and/or 6-acetylmorphine in sweat patches confirmed the use of heroin in 78.1% of the positive cases and differentiated illicit heroin use from possible ingestion of codeine or opiate-containing foods. However, the percentage of false negative results, at least in this treatment population, indicates that weekly sweat testing may be less sensitive than thrice weekly urine testing in detecting opiate use. Huestis M. A., Cone, E. J., Wong, C. J., Umbricht, A., and Preston, K. L. Journal of Analytical Toxicology, 24, pp. 509-521, 2000.

Cocaine and Metabolite Elimination Patterns in Chronic Cocaine Users During Cessation: Plasma and Saliva Analysis

It has been suggested that cocaine may accumulate in tissues following chronic administration and be detectable for an extended period of time compared to acute dosing. This study was designed to investigate the elimination patterns of cocaine and metabolites in the body fluids of chronic high-dose cocaine users. Male and female cocaine-using volunteers were housed on a closed research ward where blood and saliva specimens were collected periodically for up to 12 hours beginning immediately upon their entry to the ward. Specimens were analyzed by gas chromatography mass spectrometry for cocaine, benzoylecgonine, ecgonine methyl ester and other metabolites. On average, subjects had a history of cocaine use of approximately eight years and current cocaine use of approximately 10 days in the past 2 weeks. Time since last illicit reported use of cocaine prior to sample collection ranged from 2.5 to 63 hours. Plasma cocaine (COC) was detected in 16 subjects with an intake mean of 13.4 37.0 ng/ml (highest 162.2). The mean and highest concentration of cocaine in saliva were 19.5 41.4 ng/mL and 170.7 ng/mL, respectively. Where cocaine was detected in both plasma and saliva, the mean saliva-to-plasma (s/p) ratios was 5.2 6.1 (range 0.7 to 16.1). These data suggest that low concentrations of unmetabolized cocaine and higher concentrations of metabolites can be detected in both plasma and saliva several hours after last use. While cocaine s/p ratios were comparable to those reported from studies of acute administration, these data suggest that EME accumulates as a result of chronic administration. Moolchan, E. T., Cone, E. J., Wtsadik, A., Huestis, M. A., and Preston, K. L. Journal of Analytical Toxicology, 24, pp. 458-466, 2000.

Sweat Testing for Cocaine, Codeine and Metabolites by Gas Chromatography Mass Spectrometry

Sweat testing for drugs of abuse provides a convenient and considerably less invasive method for monitoring drug exposure than blood or urine. Numerous devices have been developed. The most common device for collection of sweat specimens in current use is the PharmChek™ Sweat Patch which usually is worn by an individual for five to ten days. This device has been utilized in several field trials comparing sweat test results to conventional urinalysis with favorable results. Two new Fast Patch devices have been developed and tested that allow rapid collection of sweat specimens. The Hand-held Fast Patch was applied to the palm of the hand and the Torso Fast Patch was applied to the abdomen. Both patches employed heat-induced sweat stimulation and a larger cellulose pad for increased drug collection. Sweat specimens were collected for 30 min at various times following administration of cocaine or codeine in controlled dosing studies and analyzed by GC-MS simultaneously for cocaine, codeine and metabolites. Cocaine and codeine were the primary analytes detected in sweat. Peak cocaine and codeine concentrations ranged from 33-3579 ng/patch and 11-1123 ng/patch, respectively, across all doses for the Hand-held Patch compared to 22-1463 ng/patch and 12-360 ng/patch, respectively for the Torso Fast Patch. Peak concentrations generally occurred 4.5-24 h after dosing and were considerably higher than those reported for the PharmChek™ Sweat Patch. Multiple mechanisms appear to be operative in determining the amount of drug and metabolite secreted in sweat including passive diffusion from blood into sweat glands and outward transdermal migration of drug. Additional important factors are the physico-chemical properties of the drug analyte, specific characteristics of the sweat collection device, site of sweat collection, and in this study, the application of heat to increase the amount of drug secreted. Huestis, M. A., Oyler, J. M., Cone, E. J., Wtsadik, A. T., Schoendorfer, D., and Joseph, R. E. Journal of Chromatography B, 733, pp. 247-264, 1999.

Tolerance to Nicotine in Nonsmokers

When administered acutely to nonsmokers, nicotine's effects on performance are inconsistent, perhaps because of suboptimal dosing or initial dysphoria that could interfere with performance. The purpose of this study was to determine if a range of nicotine doses administered for 8 days to nonsmokers would enhance psychomotor and cognitive abilities and to document the development of nicotine tolerance or sensitization. Twelve male volunteers, who reported ever smoking 5 cigarettes or less, participated in 8 consecutive experimental days in which they were administered four doses of nicotine polacrilex gum each day in this order: 0, 2, 4, and 8 mg. Performance, subjective, and physiological measures were assessed before and after each dose. Plasma nicotine concentration ranged from 6.9 to 11.5 ng/ml following the 8 mg dose. Nicotine increased rate of responding and decreased response time on working memory (digit recall); however, accuracy was impaired. Nicotine also decreased accuracy on visual scanning and attention (two-letter search), and the 8 mg dose impaired gross motor coordination (circular lights). Tolerance did not develop to the performance impairing effects of nicotine. Nicotine produced dose-related increases in ratings of dysphoria and negative mood, including tension, anxiety, nervousness, turning of stomach, and sedation. Tolerance developed to some, but not all, of these aversive effects. Tolerance also was not observed to the increased cardiovascular measures. Although tolerance developed to some of the aversive effects of nicotine, performance enhancement was not observed. These data do not support the hypothesis that nicotine-induced performance enhancement contributes to the reinforcing effects of tobacco use during the early stages of dependence development. Heishman, S. J., and Henningfield, J. E. Psychopharmacology, 152, pp. 321-333, 2000.

Development and Plasticity Section, Cellular Neurobiology Research Branch

Neuropathology of Bipolar Disorder

The literature on the neuropathology of bipolar disorder (BD) is reviewed. Postmortem findings in the areas of pathomorphology, signal transduction, neuropeptides, neurotransmitters, cell adhesion molecules, and synaptic proteins are considered. Decreased glial numbers and density in both BD and major depressive disorder (MDD) have been reported, whereas cortical neuron counts were not different in BD (in Brodmann's areas [BAs] 9 and 24). In contrast, MDD patients showed reductions in neuronal size and density (BA 9, BA 47). There are a number of findings of alterations in neuropeptides and monoamines in BD brains. Norepinephrine turnover was increased in several cortical regions and thalamus, whereas the serotonin metabolite, 5-hydroxyindoleacetic acid, and the serotonin transporter were reduced in the cortex. Several reports further implicated both cyclic adenosine monophosphate and phosphatidylinositol (PI) cascade abnormalities. G protein concentrations and activity increases were found in the occipital, prefrontal, and temporal cortices in BD. In the PI signal cascade, alterations in PKC activity were found in the prefrontal cortex. In the occipital cortex, PI hydrolysis was decreased. Two isoforms of the neural cell adhesion molecules were increased in the hippocampus of BD, whereas the synaptic protein marker, synaptophysin, was not changed. The findings of glial reduction, excess signal activity, neuropeptide abnormalities, and monoamine alterations suggest distinct imbalances in neurochemical regulation. Possible alterations in pathways involving ascending projections from the brain stem are considered. Larger numbers of BD brains are needed to further refine the conceptual models that have been proposed, and to develop coherent models of the pathophysiology of BD. Vawter, M.P., Freed, W.J., and Kleinman, J.E. Biological Psychiatry, 48, pp. 486-504, 2000.

Kinetics of Recovery at Mu Opioid Receptors

To investigate a previous observation that classical antagonists behave as agonists at mutant H297N and H297Q mu opioid receptors, we compared the kinetics of recovery from opioids at wild-type and mutant mu receptors expressed in voltage-clamped Xenopus oocytes. The cDNA for the potassium channel GIRK1 was coinjected into the oocytes with that of the mu receptors to transduce agonist binding into a coupled electrophysiological response. The kinetics of recovery were estimated by brief test pulses of the agonist normorphine given at a frequency of 0.67 or 1 per min. After treatment with a variety of agonists, the receptors recovered from desensitization at rates that depended on the agonist, but there was little difference between mutant and wild-type receptors. Antagonists, however, induced agonist-like currents and demonstrated faster recovery at the mutant receptors. These results suggest that His-297 may comprise part of an antagonist subsite. This conclusion, when coupled with the steric theory that intrinsic activity depends on independent binary equilibration of a drug between agonist and antagonist subsites, could unify the paired observations that antagonists become agonists and recover faster at the mutant than at the wild-type receptors. Spivak, C.E. and Beglan, C.L. Synapse, 38, pp. 254-260, 2000.

Dysregulation of the Neural Cell Adhesion Molecule and Neuropsychiatric Disorders

Cell adhesion molecule proteins play a diverse role in neural development, signal transduction, structural linkages to extracellular and intracellular proteins, synaptic stabilization, neurogenesis, and learning. Three basic mRNA isoforms and potent posttranslational modifications differentially regulate these neurobiological properties of the neural cell adhesion molecule (N-CAM). Abnormal concentrations of N-CAM 105-115 kDa (cN-CAM), N-CAM variable alternative spliced exon (VASE), and N-CAM secreted exon (SEC) are related to schizophrenia and bipolar neuropsychiatric disorders. These N-CAM isoforms provide potential mechanisms for expression of multiple neurobiological alterations between controls and individuals with schizophrenia or bipolar illness. Multiple processes can trigger the dysregulation of N-CAM isoforms. Differences in neuropil volume, neuronal diameter, gray matter thickness, and ventricular size can be related to N-CAM neurobiological properties in neuropsychiatric disorders. Potential test of the N-CAM dysregulation hypothesis of neuropsychiatric disorder is whether ongoing dysregulation of N-CAM would cause cognitive impairments, increased lateral ventricle volume, and decreased hippocampal volume observed in schizophrenia and to a lesser extent in bipolar disorder. An indirect test of this theory conducted in animal experiments lend support to this N-CAM hypothesis. N-CAM dysregulation is consistent with a synaptic abnormality that could underlie the disconnection between brain regions consistent with neuroimaging reports. Synapse stability and plasticity may be part of the molecular neuropathology of these disorders. Vawter, M.P. European Journal of Pharmacology 405, pp. 385-395, 2000.

Molecular Neuropsychiatry Section, Cellular Neurobiology Research Branch

Methamphetamine-induced Neurotoxicity is Attenuated in Transgenic Mice with a Null Mutation for Interleukin-6

Increasing evidence implicates apoptosis as a major mechanism of cell death in methamphetamine (METH) neurotoxicity. The involvement of a neuroimmune component in apoptotic cell death after injury or chemical damage suggests that cytokines may play a role in METH effects. In the present study, IRP investigators examined if the absence of IL-6 in knockout (IL-6-/-) mice could provide protection against METH-induced neurotoxicity. Administration of METH resulted in a significant reduction of [(125)I]RTI-121-labeled dopamine transporters in the caudate-putamen (CPu) and cortex as well as depletion of dopamine in the CPu and frontal cortex of wild-type mice. However, these METH-induced effects were significantly attenuated in IL-6-/- animals. METH also caused a decrease in serotonin levels in the CPu and hippocampus of wild-type mice, but no reduction was observed in IL-6-/- animals. Moreover, METH induced decreases in [(125)I]RTI-55-labeled serotonin transporters in the hippocampal CA3 region and in the substantia nigra-reticulata but increases in serotonin transporters in the CPu and cingulate cortex in wild-type animals, all of which were attenuated in IL-6-/- mice. Additionally, METH caused increased gliosis in the CPu and cortices of wild-type mice as measured by [(3)H]PK-11195 binding; this gliotic response was almost completely inhibited in IL-6-/- animals. There was also significant protection against METH-induced DNA fragmentation, measured by the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeled (TUNEL) cells in the cortices. The protective effects against METH toxicity observed in the IL-6-/- mice were not caused by differences in temperature elevation or in METH accumulation in wild-type and mutant animals. Therefore, these observations support the proposition that IL-6 may play an important role in the neurotoxicity of METH. Ladenheim, B., Krasnova, I. N., Deng, X., Oyler, J. M., Polettini, A., Moran, T. H., Huestis, M. A., and Cadet, J. L. Molecular Pharmacology, 58, pp. 1247-1256, 2000.

Methamphetamine-induced Apoptosis is Attenuated in the Striata of Copper-Zinc Superoxide Dismutase Transgenic Mice

Administration of methamphetamine caused significant increases in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells, in poly (ADP-ribose) polymerase (PARP) cleavage, as well as in caspase-3 activity in the striata of C57BL/6J mice. In contrast, all these effects were markedly suppressed in the copper-zinc superoxide dismutase transgenic mice. These results indicate that superoxide radicals might be important factors in METH-induced cell death. Deng, X. and Cadet, J. L. Brain Research Molecular Brain Research, 83, pp. 121-124, 2000.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Laboratory

Self-administration Behavior is Maintained by the Psychoactive Ingredient of Marijuana in Squirrel Monkeys

Most drugs abused by humans are intravenously self-administered by experimental animals. The lack of such evidence with marijuana (cannabis) or its psychoactive ingredient THC has led some to assume that marijuana is less addicting and dangerous than other "hard" drugs such as crack cocaine or heroin. We have now demonstrated that THC is actively and persistently self-administered by squirrel monkeys. The THC self-administration behavior was comparable in intensity to that maintained by cocaine in a control group of monkeys under identical conditions and was obtained using a range of doses in agreement with the single doses self-administered by humans smoking marijuana cigarettes. Also, treatment with a drug (SR141716A) that blocks the action of THC at cannabinoid receptors in the brain (CB 1 receptors), almost completely eliminated THC self-administration behavior but did not reduce cocaine self-administration behavior. The methodology provides an exciting opportunity for studying brain mechanisms involved in cannabinoid self-administration behavior. It also provides an exciting opportunity to study possible therapeutic strategies for treating marijuana abuse and to develop drugs possessing therapeutic efficacy similar to or better than marijuana or THC but lacking their potential for abuse. Tanda, G., Munzar, P., and Goldberg, S. R. Nature Neuroscience, 3, pp. 1073-1074, 2000.

Clinical Psychopharmacology Section, Medications Discovery Research Branch

Evidence for Possible Involvement of 5-HT2B Receptors in the Cardiac Valvulopathy Associated with Fenfluramine and other Serotonergic Medications

Serotonergic medications with various mechanisms of action are used to treat psychiatric disorders and are being investigated as treatments for drug dependence. The occurrence of fenfluramine-associated valvular heart disease (VHD) has raised concerns that other serotonergic medications might also increase the risk of developing VHD. We hypothesized that fenfluramine or its metabolite, norfenfluramine, and other medications known to produce VHD, have preferentially high affinities for a particular serotonin receptor subtype capable of stimulating cell growth on heart valves. Medications known or suspected to cause VHD (positive controls) and medications not associated with VHD (negative controls) were screened for activity at eleven cloned serotonin receptor subtypes using ligand binding methods and functional assays. The positive control drugs were ()-fenfluramine, (+)-fenfluramine, (-)-fenfluramine, its metabolites ()-norfenfluramine, (+)-norfenfluramine, (-)-norfenfluramine, ergotamine, methysergide and its metabolite, methylergonovine. The negative control drugs were phentermine, fluoxetine, its metabolite, norfluoxetine, trazodone and its active metabolite, m-chlorophenylpiperazine (mCPP). ()-, (+)- and (-)-Norfenfluramine, ergotamine and methylergonovine all had preferentially high affinities for the cloned human serotonin 5-HT2B receptor and stimulated the 5-HT2B receptor. Our data imply that activation of 5-HT2B receptors is necessary to produce VHD and that serotonergic medications which do not activate 5-HT2B receptors are unlikely to produce VHD. We suggest that all clinically available medications with serotonergic activity as well as their active metabolites be screened for agonist activity at 5-HT2B receptors and that clinicians consider suspending their use of medications with significant activity at 5-HT2B receptors. Rothman, R.B., Baumann, M.H., Savage, J.E., Rauser, L., McBride, A., Hufisein, S., and Roth, B.L. Circulation, 102, pp. 2836-2841, 2000.

Amphetamine-type Central Nervous System Stimulants Release Norepinephrine More Potently than they Release Dopamine and Serotonin

A large body of evidence supports the hypothesis that mesolimbic dopamine (DA) mediates, in animal models, the reinforcing effects of central nervous system stimulants such as cocaine and amphetamine. The role DA plays in mediating amphetamine-type subjective effects of stimulants in humans remains to be established. Both amphetamine and cocaine increase norepinephrine (NE) via stimulation of release and inhibition of reuptake, respectively. If increases in NE mediate amphetamine-type subjective effects of stimulants in humans, then one would predict that stimulant medications that produce amphetamine-type subjective effects in humans should share the ability to increase NE. To test this hypothesis, we determined, using in vitro methods, the neurochemical mechanism of action of amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), (+)-methamphetamine, ephedrine, phentermine, and aminorex. As expected, their rank order of potency for DA release was similar to their rank order of potency in published self-administration studies. Interestingly, the results demonstrated that the most potent effect of these stimulants is to release NE. Importantly, the oral dose of these stimulants, which produce amphetamine-type subjective effects in humans, correlated with their potency in releasing NE, not DA, and did not decrease plasma prolactin, an effect mediated by DA release. These results suggest that NE may contribute to the amphetamine-type subjective effects of stimulants in humans. Rothman, R.B., Baumann, M.H., Dersch, C.M., Romero, D.V., Rice, K.C., Carroll, F.I. and Partilla, J.S. Synapse, 39, pp. 32-41, 2001.

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