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National Institute on Drug Abuse

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

February, 2000

Research Findings

Intramural Research

Treatment Section, Clinical Pharmacology and Therapeutics Research Branch

Low-Dose Oral Cocaine in Humans: Acquisition of Discrimination and Time Course of Effects

As a tool for initial human screening of new anti-addictive compounds, oral cocaine may have practical advantages over intravenous, intranasal, or smoked cocaine. However, the nature and extent of its effects need further characterization. We tested the discriminability, subjective effects, and cardiovascular effects of 50 mg oral cocaine vs. placebo in seven cocaine-abusing volunteers across 50 two-hour sessions. Our behavioral-testing procedure permitted precise measurement of the time course of drug effects within sessions. Oral cocaine's effects began at a longer latency than has been reported for other routes, and had a longer duration. Six out of seven participants acquired the discrimination (though this typically required 15 to 30 sessions). In most participants, oral cocaine produced increases in ratings of "liking," "alertness," and "good effects," and in motor performance. Nonetheless, for most participants, peak heart rate and blood pressure remained within the range seen with placebo. Additional testing with one participant suggested that oral cocaine was discriminable and reinforcing at 25 mg and possibly 12.5 mg. The results support the discriminability of oral cocaine (and the sensitivity of the behavioral-testing procedure used). However, there were some intriguing dissociations between oral cocaine's discriminability and its identifiability. Epstein, D., Silverman, K., Henningfield, J.E., and Preston, K.L. Behavioural Pharmacology, 10, pp. 531-542, 1999.

Naltrexone Shortened Opioid Detoxification with Buprenorphine

In a double-blind, randomized clinical trial we evaluated the impact on withdrawal symptoms of combining naltrexone to a 4-day sublingual buprenorphine taper for short opioid detoxification. The experimental group (NB; n = 32) received placebo on day 1 and escalating doses of oral naltrexone on days 2-8; the control group (PB; n = 28) received placebo on days 1-7 and naltrexone on day 8. Main outcome measures were observer-rated withdrawal and use of medications for opioid withdrawal. Most (59%) patients in the NB group experienced clinically relevant withdrawal on day 2, but none after day 5. In contrast, withdrawal increased over time in the PB group. Withdrawal signs precipitated by the first naltrexone dose were similar in intensity whether on day 2 of the buprenorphine taper (NB) or on day 8 (4 days after the last buprenorphine dose; PB). However, 7 patients in the NB group dropped out on day 2, while only one patient dropped out in the PB group on day 8. Both groups received similar amounts of adjunct medication, with only 25% requiring sedatives. Initiating naltrexone on day 2 appears to abolish withdrawal symptoms after day 5, thus shortening the duration of withdrawal symptoms, and peak withdrawal was of moderate intensity. Thus, naltrexone combined with buprenorphine is an acceptable and safe procedure for shortened opioid detoxification and induction of naltrexone maintenance. Umbricht, A., Montoya, I.D., Hoover, D.R., Demuth, K.L., Chiang, C.-T., and Preston, K.L. Drug and Alcohol Dependence, 57, pp. 181-190, 1999.

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

Sweat and urine specimens were collected from methadone maintenance patients to evaluate sweat testing to monitor cocaine use. Paired sweat patches worn for one week; urine specimens collected three times weekly. All patches (N = 930) were analyzed by ELISA immunoassay (cutoff concentration 10 ng/mL); a subset (N = 591) were also analyzed by GC-MS for cocaine, benzoylecgonine (BZE), and ecgonine methyl ester (EME) (cutoff concentration 5 ng/mL). Urine specimens were analyzed qualitatively (cutoff 300 ng/mL), and subsets were analyzed semiquantitatively (LOD = 30 ng/mL) and by GC-MS for cocaine (LOD = 5 ng/mL). Results were evaluated to determine the relative amounts of cocaine and its metabolites in sweat, assess replicability in duplicate patches, compare ELISA and GC-MS analyses in sweat, and compare the detection of cocaine in sweat and urine. Cocaine was detected by GC-MS in 99% of ELISA positive sweat patches; median concentrations of cocaine, BZE and EME were 378, 78.7 and 74 ng/mL. Agreement in duplicate patches was approximately 90% by ELISA analysis. Sensitivity, specificity and efficiency of sweat ELISA cocaine results as compared to sweat GC-MS results were all between 91 and 94%. Sensitivity, specificity and efficiency between ELISA sweat patch and qualitative urine results were 97.6%, 60.5%, and 77.7%. These results support the utility of sweat patches for monitoring cocaine use. Preston, K.L., Huestis, M.A., Wong, C.J., Umbricht, A., Goldberger, B.A., and Cone, E.J. Journal of Analytical Toxicology, 23, pp. 313-322, 1999.

Chemistry & Drug Metabolism Section, Clinical Pharmacology and Therapeutics Branch

Selective Deficits in Reflective Cognition of Polydrug Abusers

Reflective cognitive processing has been shown to be impaired in detoxified alcoholics who were otherwise cognitively intact. The goal of this study was to determine if substance abusers exhibited similar specific cognitive deficits. Fifteen subjects with histories of polydrug abuse and 15 normal control subjects were administered a series of cognitive tests. The tests assessed various learning and memory functions, including explicit and implicit memory, meta-cognition, working memory, memory consolidation, retrieval of information from long-term memory, and access to knowledge in long-term memory. Substance abusers did not differ from controls with respect to most cognitive domains, especially under conditions of stimulus-driven processing. However, substance abusers were impaired in using previously acquired knowledge and implicit memory on unstructured processing tests (fragmented pictures) and in inhibiting intrusion errors in word recall. These deficits in reflective functioning were negatively correlated with education and were most evident in subjects who did not graduate from high school. Deficits in reflective functioning are associated with drug-seeking and drug-taking behaviors and may be a risk factor for the initiation or maintenance of substance use disorders. Heishman, S.J., Weingartner, H.J., and Henningfield, J.E. Psychology of Addictive Behaviors, 13, pp. 227-231, 1999.

Medicinal Chemistry Section, Medications Discovery Research Branch

2D and 3D QSAR Modeling of Dopamine Transporter Ligands

We have synthesized >100 analogs of benztropine as novel probes for the dopamine transporter. Many of these compounds demonstrate high affinity and selectivity for the dopamine transporter and yet do not share a behavioral profile with cocaine in animal models of psychostimulant abuse. We have hypothesized that these compounds may be interacting at a binding domain on the dopamine transporter that is distinct from cocaine and that this may, in part, be related to their distinctive behavioral profile. This suggests that it may be possible to design dopamine uptake inhibitors that have therapeutic value for cocaine abuse treatment without inherent abuse potential. In order to study further the relationship of the structures of these compounds to their binding domain, we have begun to implement several molecular modeling techniques. Comparative Molecular Field Analysis (COMFA) derives the relationship between 3D-structures with binding affinities and provides a steric and electronic description of the binding domain of these ligands at the dopamine transporter. The contour maps that have been derived from the benztropines do indeed differ from those derived for cocaine and its analogs. Furthermore, these studies have confirmed structure-activity relationships previously derived in these series of compounds and now provide highly predictive models (q2=0.78) with which new ligands can be designed. In addition, a 2D QSAR model has been derived using Genetic Algorithm Partial Least Squares methodology. This highly predictive model (q2=0.88) has been used to screen the NCI database and a number of potential new leads with structural diversity have been discovered. It is anticipated that these studies will lead to the design of novel probes for the dopamine transporter and may provide clues toward a cocaine abuse therapeutic. Newman, A.H., Robarge, M., Izenwasser, S., and Kline, R.H. Journal of Medicinal Chemistry, 42, pp. 3502-3509, 1999.

Clinical Psychopharmacology Section, Medications Discovery Research Branch

GBR12909 Attenuates Amphetamine-induced Striatal Dopamine Release as Measured by [11C]raclopride Continuous Infusion PET Scans

Major neurochemical effects of methamphetamine include release of dopamine (DA), serotonin (5-HT) and norepinephrine (NE) via a carrier-mediated exchange mechanism. Preclinical research supports the hypothesis that elevations of mesolimbic DA mediate the addictive and reinforcing effects of methamphetamine and amphetamine. The present study determined the ability of GBR12909 to attentuate amphetamine-induced increases in striatal DA as measured by [11C]raclopride continuous infusion PET scans in two Papio Anubis baboons. [11C]Raclopride was given in a continuous infusion paradigm. A 1.5 mg/kg amphetamine i.v. bolus was administered which caused a significant (30.3%) reduction in the volume of distribution (V3). The percent reduction in the volume of distribution and hence a measure of the intrasynaptic DA release ranged between 22 and 41%. GBR12909 (1 mg/kg, slow i.v. infusion) was administered 90 min before the administration of the radiotracer. GBR12909 inhibited amphetamine-induced dopamine release by 74%. Previous studies showed that 1 mg/kg GBR12909 occupies about 20% of DA transporters in the brain. The present results demonstrate that relatively low DA transporter occupancies by GBR12909 can substantially reduce amphetamine-induced DA release. These occupancy levels are achievable in humans administered GBR12909 via the oral route. Thus, these experiments suggest that GBR12909 is an important prototypical medication to test the hypothesis that stimulant-induced euphoria is mediated by DA, and, if the DA hypothesis is correct, a potential treatment agent for cocaine and methamphetamine abuse. Villemagne, V.L., Wong, D.F., Yokoi, F., Stephane, M., Rice, K.C., Matecka, D., Clough, D.J., Dannals, R.F., and Rothman, R.B. Synapse, 33, pp. 268-273, 1999.

Molecular Neuropsychiatry Section, Cellular Neurobiology Branch

Reversal by Delta Opioid Peptide, DADLE, of the Striatal Dopaminergic Damage Caused by Methamphetamine

Methamphetamine (METH) has been known to cause striatal dopaminergic (DA) damage. We have shown in the past that a pretreatment of delta opioid peptide DADLE can block the striatal DA damage caused by METH. Considering the potential clinical relevance of the use of DADLE, it would be of interest to examine if DADLE can "reverse" a pre-existing brain damage caused by METH. Two weeks after METH administration, a 75% loss of DA transporters was found in the striatum of CD-1 mice. An injection of DADLE, on day 14 after the METH treatment, was able to restore the DA transporter level back to the control. These results confirm the neuroprotective property of DADLE and suggest a potential use of DADLE in treating psychostimulant-induced brain damage. Tsao, L.-I., Cadet, J.L. and Su, T.-P. European Journal of Pharmacology, 372, pp. R5-R7, 1999.

Molecular Neuropsychiatry Section, Cellular Neurobiology Branch

Null Mutation of c-Fos Causes Exacerbation of Methamphetamine-induced Neurotoxicity

Methamphetamine neurotoxicity has been demonstrated in rodents and nonhuman primates. These neurotoxic effects may be associated with mechanisms involved in oxidative stress and the activation of immediate early genes (IEG). It is not clear, however, whether these IEG responses are involved in a methamphetamine-induced toxic cascade or in protective mechanisms against the deleterious effects of the drug. As a first step toward clarifying this issue further, the present study was thus undertaken to assess the toxic effects of methamphetamine in heterozygous and homozygous c-fos knockout as well as wild-type mice. Administration of methamphetamine caused significant reduction in [(125)I]RTI-121-labeled dopamine uptake sites, dopamine transporter protein, and tyrosine hydroxylase-like immunohistochemistry in the striata of wild-type mice. These decreases were significantly exacerbated in heterozygous and homozygous c-fos knockout mice, with the homozygous showing greater loss of striatal dopaminergic markers. Moreover, in comparison with wild-type animals, both genotypes of c-fos knockout mice showed more DNA fragmentation, measured by the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeled nondopaminergic cells in their cortices and striata. In contrast, wild- type mice treated with methamphetamine demonstrated a greater number of glial fibrillary acidic protein-positive cells than did c-fos knockout mice. These data suggest that c-fos induction in response to toxic doses of methamphetamine might be involved in protective mechanisms against this drug-induced neurotoxicity. Deng X., Ladenheim B., Tsao L., and Cadet J.L., Journal of Neuroscience, 19, pp. 10107-10115, 1999.

Dual Mechanism of Fas-induced Cell Death in Neuroglioma Cells: a Role for Reactive Oxygen Species

ApoI/Fas belongs to the tumor necrosis factor receptor (TNFR) superfamily and mediates cell death in various cell types. A dual mode of Fas-triggered cell death has been reported depending on cell types used in the experiments. The present study was carried out to test the possible role of reactive oxygen species in this dual mechanism in neuroglioma cells. Anti-Fas antibody caused dose-dependent and time- dependent increases in cell death measured by lactate dehydrogenase (LDH) release in control neuroglioma cells and in cells that were transfected with catalase cDNA. However, cells transfected with copper/zinc superoxide dismutase (Cu/ZnSOD) cDNA showed marked attenuation of Fas-induced LDH release. Moreover, flow cytometry and confocal microscopy revealed that Fas-induced cell death in control cells occur mostly through an apoptotic process. This process was also completely abrogated in cells overexpressing catalase or copper/zinc superoxide dismutase (Cu/ZnSOD). Further experiments revealed that Fas-induced cell death was associated with increased formation of Superoxide anions in control neuroglioma cells and in cells overexpressing catalase. Increases were significantly suppressed by Cu/ZnSOD overexpression. These data indicate that Fas-mediated cell death in neuroglioma cells occur, in part, through the production of reactive oxygen species (ROS). These observations also suggest that Fas-induced cell death in these cells occur through apoptosis and necrosis. Thus overexpression of Cu/ZnSOD caused the suppression of both types of Fas-induced cell death whereas catalase prevented apoptotic but not necrotic cell death. These observations are discussed in terms of their support for a role for both peroxides and superoxide radicals in Fas- induced cell death. Jayanthi, S., Ordonez, S., McCoy, M.T., and Cadet, J.L. Molecular Brain Research, 72, pp. 158-165, 1999.

Differential Gene Expression in Methamphetamine Neurotoxicity Identified Using cDNA Arrays

Methamphetamine (METH) can cause damage to monoaminergic systems in mammals. A role for oxygen-based radicals in the neurotoxic effects of the drug is well supported by the accumulated literature. Thus far, however, very little is know about the contribution of various genes to the neurotoxic effects of METH. Using cDNA microarray technology, we have monitored the expression pattern of 196 stress- and apoptotic-related genes. MRNA for microarray hybridization was obtained from different brain regions of mice treated with a neurotoxic dose of METH and compared to saline-treated controls. The results show differential cortical expression of glutathione S-transferase; MmRad51; FAF1; Adenosine A1M receptor; T-lymphocyte activation gene; Vav; GDP-GTP exchange factor; Golgi-4-transmembrane spanning transporter and IRF1. Experiments are underway using other brain regions. This approach will be valuable for identifying genes that contribute to the neurotoxicity of METH and of other agents that cause neurodegenerative disorders. Jayanthi, S., Krasnova, I., Ladenheim, B., and Cadet, J.L., Poster, 1999. Society for Neuroscience Annual Meeting, Miami, FL, October 23-29, 1999.

Toxic Effects of Methamphetamine and Dopamine in Cell Culture

Methamphetamine (METH) and dopamine (DA) can cause neurotoxic damage both in vitro and in vivo. The mechanisms of action involve the increased production of free radicals. The present study was undertaken to compare and contrast the toxic effects of METH and DA by using an immortalized neural cell line. Both METH and DA caused dose-dependent increase in production of reactive oxygen species (ROS) and cell death. Cell death caused by these agents was characterized by cytoplasmic vacuolar formation, shrinkage of cytoplasm and nuclear dissolution. Flow cytometric evaluation also revealed that these toxins cause changes similar to those observed in cells undergoing apoptosis. Furthermore, DNA electrophoresis showed that both METH and DA induced DNA ladder formation. When taken together these observations suggest that METH and DA cause these cells to die via apoptosis. Further experiments indicated that growth of these cells in low (1%) serum or in the absence of serum markedly enhanced the apoptotic effects of both drugs. These data provide further support for the idea that both METH and DA can cause ROS-mediated apoptosis. Cadet, J.L., Ordonez, S. and Burrell, S., Poster, 1999. Oxygen Society Meeting, New Orleans, LA, November 17-22, 1999.

Neuroimaging Research Branch

[Br-76]BAP is a Novel Ligand for Imaging nAChRs

5-[Br-76]Bromo-3-[[2(S)-azetidinyl] methoxy]pyridine ([Br-76]BAP), a novel nicotinic acetylcholine receptor ligand, was synthesized using [Br-76]bromide and evaluated as a potential radiotracer for use with positron emission tomography (PET). The radiochemical yield was 25%, and the specific radioactivity was on the order of 1 Ci/micromol. In vitro studies using rat cortical and thalamic membranes demonstrated >90% of maximal specific [Br-76]BAP binding after 60 min. For cortical and thalamic membranes, the binding affinities (Kd) were 36 +/- 9 and 30 +/- 9 pM respectively. Although data were best fit by a single population of binding sites, Scatchard plots were nonlinear, and the Hill coefficients were <1, suggesting the presence of a lower-affinity binding site. Maximal binding site density (Bmax) values were 90 +/- 17 and 207 +/- 33 fmol/mg in the cortex and thalamus, respectively. In vitro and ex vivo autoradiography showed binding of [Br-76]BAP was high in the thalamus and presubiculum, moderate in the cortex and striatum, and low in the cerebellum and hippocampus. in vivo binding of [Br-76]BAP in whole rat brain was blocked by the preinjection of (S)(-)-nicotine (0.3 mg/kg) by 91% at 300 min. In PET studies using Rhesus monkeys, radiotracer accumulation was highest in the thalamus, and cytisine and nicotine effectively displaced thalamic radioactivity by 60% and by 50%, respectively. These results indicate that [Br-76]BAP is a promising radioligand for characterizing nicotinic acetylcholine receptors in vivo using PET. Sihver, W., Fasth, K.J., Horti, A.G., Koren, A.O., Bergstrom, M., Lu, L., Hagberg, G., Lundqvist, H., Dannals, R.F., London, E.D., Nordberg, A., and Langstrom, B. J. Neurochem. 73(3), pp. 1264-72, 1999.

2-[18F]F-A-83580: A PET Radioligand for alpha4beta2 _Nicotinic Acetylcholine Receptors

Until recently noninvasive in vivo studies of central nicotinic acetylcholine receptors (nAChRs) have been impeded by the absence of appropriate radiotracers. Taking into account that central nAChRs have been implicated in a variety of brain functions, including cognitive processes, and also are affected in various pathological conditions, such as Alzheimer's and Parkinson's diseases, noninvasive imaging techniques such as PET, could be powerful tools for studying these receptors in vivo. A new compound for the alpha4beta2 subtype of nAChRs, 2-fluoro-A-85380, synthesized and labeled with F-18 by the NIDA radiochemistry group, has been evaluated in in vitro binding assays with membranes of rat brain and in vivo by PET in Rhesus monkey. The ligand has high affinity for alpha4beta2 nAChRs (Kd = 50 n), crosses the blood-brain barrier and distributes in brain regions known to contain high densities of alpha4beta2 nAChRs. A high level of specific binding was achieved 4 h after administration, as indicated by the thalamus-cerebellum/cerebellum radioactivity ratio of 3.3. The specificity of binding was confirmed by displacement study with cytisine. These results, in combination with data demonstrating low toxicity of 2-[F-18]F-A-85380, indicate that this radiotracer is an excellent candidate for PET imaging of central nAChRs in human subjects. Chefer, S.I., Horti, A.G., Koren, A.O., Gundisch, D., Links, J.M., Kurian, V., Dannals, R.F., Mukhin, A.G., and London, E.D. NeuroReport, 10, pp. 2715-2721, 1999.

Comparison Study of [C-11]iomazenil and [I-123]iomazenil by PET and SPECT

Although PET and single photon emission computed tomography (SPECT) are increasingly used for quantitation of neuroreceptor binding, almost no studies to date have involved a direct comparison of the two imaging techniques. One study found a high level of agreement between the two techniques, although there was a systematic 30% increase in measures of benzodiazepine receptor binding in SPECT compared with PET. The purpose of the current study was to directly compare quantitation of benzodiazepine receptor binding in the same human subjects using PET and SPECT with high specific activity [C-11]iomazenil and [I-123]iomazenil, respectively. All subjects were administered a single bolus of high specific activity iomazenil labeled with C-11 or I-123 followed by dynamic PET or SPECT imaging of the brain. Arterial blood samples were obtained for measurement of metabolite-corrected radioligand in plasma. Compartmental modeling was used to fit values for kinetic rate constants of transfer of radioligand between plasma and brain compartments. These values were used to calculate binding potential (BP=Bmax/Kd) and the fraction of free non-protein-bound parent compound (V3'). Mean values for V3' in PET and SPECT were as follows: temporal cortex 235 and 223 ml/g, frontal cortex 236 and 223 ml/g, occipital cortex 283 and 315 ml/g, and striatum 44 and 74 ml/g. These preliminary findings indicate that PET and SPECT provide comparable results in quantitation of neuroreceptor binding in the human brain. Bremner, J.D., Baldwin, R., Horti, A., Staib, L.H., Ng, C.K., Tan, P.Z., Zea-Ponce, Y., Zoghbi, S., Seibyl, J.P., Soufer, R., Charney, D.S., and Innis, R.B. Psychiatry Res.: Neuroimaging, 91, pp. 79-91, 1999.

Methylphenidate and Neuroimaging

In June 1995 a research article appeared in the Archives of General Psychiatry (Volkow et al. 1995) and asked the provocative question "Is methylphenidate like cocaine?". This question was addressed by examining the cerebral location and time course of the effects of these drugs using functional neuroimaging. The answer is not simple. Although these drugs have similar mechanisms of action, there are more subtle differences that make methylphenidate a safe and effective medication for the treatment of attention-deficit hyperactivity disorder, and cocaine an addictive and dangerous agent. To assist in the interpretation of brain imaging literature, this review addresses briefly the pharmacology of cocaine and methylphenidate, the basic principles of brain imaging techniques, and the strategies used to map and quantify the effects of pharmacological agents in the living human brain. Ernst, M., Earle, A., and Zametkin, A.J. In: Ritalin: Theory and Practice, Laurence L. Greenhill and Betty Osman, eds, Mary Ann Liebert, Inc. Publishers, pp. 375-384, 1999.

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