National Institute on Drug Abuse
Director's Report to the National Advisory Council on Drug Abuse
Office of the Director, IRP
During the past year, Intramural Research Program Director Dr. Barry Hoffer's work has focussed on effects of trophic peptides in the GDNF family, and on regulation of GDNF-family receptors. This work, carried out primarily in collaborating laboratories during the past year, will move to DIR labs this summer. In addition, work will be initiated at DIR on Nurr-1, which appears to be a critical factor for the specific development of midbrain DA neurons. New initiatives to be developed during the coming year involve multiunit recording approaches and ultrastructural studies, as well as the electrochemical, electrophysiological, and immunocytochemical protocols traditionally used by this laboratory.
Brain Imaging Section, Neuroscience Branch
Global Brain Glucose Metabolism is not Confounded by Modest Changes in Plasma Glucose following Acute Alpha2 Blockade by Idazoxan
The sympathetic nervous system can modulate glucose levels via several mechanisms, including inhibition of insulin release by alpha2-adrenergic receptors. Such effects could potentially confound measurements of brain glucose metabolism during studies of the central actions of sympathomimetic drugs. To test this hypothesis, plasma glucose, insulin and sympathetic responses following infusion of idazoxan, a selective alpha2 antagonist, or placebo were compared with global brain metabolism obtained from positron emission tomography (PET) scans using [18F]-fluoro-deoxyglucose before and after the infusion. Glucose levels fell and fractional levels of insulin rose after idazoxan, compared to placebo. Relative increases in insulin correlated with increases in epinephrine after active drug, consistent with the role of alpha2-adrenoreceptors in regulating insulin release. Estimates of global brain glucose metabolism did not appear to be influenced by the modest changes in plasma glucose. Schmidt, M.E., Goldstein, D.S., Schouten, J.L., Matochik, J.A., Kim, H.G., Potter, W.Z. Acute Alpha2 Blockade by Idazoxan Increases Insulin and Lowers Plasma Glucose During Positron Emission Tomography. Psychopharmacol Bull, 33, pp. 253-259, 1997.
Anxiety Level Affects Cerebral Glucose Metabolism During Repeated PET Trials
Cerebral glucose utilization was higher during the first positron emission tomography (PET) session than during the second PET session, as assayed using the [18F]fluorodeoxyglucose method in male human volunteers. This difference was due largely to data from subjects with low Trait Anxiety, since subjects with high anxiety showed similar metabolism in both PET sessions. High-anxiety subjects showed greater right/left ratios of cerebral metabolism than low-anxiety subjects, particularly during the second PET session. These findings suggest that the level of anxiety may be an important variable to consider in PET studies using multiple sessions. Stapleton, J.M., Morgan, J.M., Liu, X., Yung, B.C.-K., Phillips, R.L., Wong, D.F., Shaya, E.K., Dannals, R.F., and London, E.D. Cerebral Glucose Utilization is Reduced in Second Test Session. J. Cereb. Blood Flow Metab. 17, pp. 704-712, 1997.
I-IPH: A Promising Epibatidine Analogue for Imaging Neuronal Nicotinic Receptors
An analog of epibatidine (EB) was synthesized with an iodine atom in the 2 position of the pyridyl ring. This analog, (+/-)-exo-2-(2-iodo-5-pyridyl)-7-azabicyclo[2.2.1]heptane
(IPH), as well as its two stereoisomers, displayed high affinity for neuronal nicotinic acetylcholine receptors (nAChRs). Therefore, radioiodinated IPH, [125I]IPH, was synthesized with specific radioactivities consistently > 1000 Ci/mmol, and its properties as a radioligand for neuronal nAChRs were evaluated. The characteristics of [125I]IPH binding in tissue homogenates appeared to be virtually identical to those reported for [3H]epibatidine binding, but the high specific radioactivity of [125I]IPH greatly facilitated measurements of nicotinic receptors in tissues with relatively low receptor densities and/or where tissues are in limited supply. Autoradiography with [125I]IPH provided clear localization of nAChRs in brain and adrenal gland after film exposure times of < or = 2 days. Investigators conclude that [125I]IPH will be a very useful radioligand for the study of neuronal nicotinic receptors in brain and in peripheral ganglia. Davila-Garcia, M.I., Musachio, J.L., Perry, D.C., Xiao, Y., Horti, A. London, E.D., Dannals, R.F., and Kellar K.J. [125I]IPH, An Epibatidine Analog, Binds with High Affinity to Neuronal Nicotinic Cholinergic Receptors. J. Pharmacol. Exp. Ther. 282, pp. 445-451, 1997.
Molecular Neuropsychiatry Section, Neuroscience Branch
Evidence that Methamphetamine Damages Human DNA
Immortalized neural cells obtained from rat mesencephalon were used to further assess the role of oxidative stress in methamphetamine (METH)-induced neurotoxicity. Investigators tested if the anti-death proto-oncogene, Bcl-2, could protect against METH-induced cytotoxicity. Results suggested that METH can cause cell death in a manner that resembles apoptosis. This was demonstrated by flow cytometry, fluorescence microscopy and DNA gel electrophoreses. Bcl2 protected cells against METH-induced molecular changes. These results are the first to demonstrate the possibility that METH use might cause DNA damage in humans who abuse that drug. Cadet, J.L., Ordonez, S.V., and Ordonez, J.V. Methamphetamine Induces Apoptosis in Immortalized Neural Cells: Protection by the Protoncogene, Bcl2. Synapse, 25, pp. 176-184, 1997.
Increased EEG Beta Activity is Manifested During Cocaine Withdrawal
This investigation shows that the brains of chronic cocaine users have increased beta activity on the EEG. Together with the demonstration of that these subjects have neuropsychological impairment, these data indicate that chronic cocaine abuse results in a brain disorder that can be characterized as a neuropsychiatric syndrome. Herning, R.I., Guo, X., Better, W.E., Weinhold, L.L., Lange, W.R., Cadet, J.L., and Gorelick, D.A. Neurophysiological Signs in Cocaine Dependence: Increased EEG Beta During Withdrawal. Biol. Psychiatry, 41, pp. 1087-1094, 1997.
Molecular Neuroadaptive Changes in the Dopamine Transporter and Cocaine Addiction
Binding of cocaine to dopamine transporters leads to inhibition of dopamine reuptake and plays a pivotal role in its addictive effects. In contrast to cocaine, clinically used dopamine reuptake inhibitors are considered not to possess abuse potential. One factor that may underlie differences between cocaine and other dopamine reuptake inhibitors is pharmacological heterogeneity among dopamine reuptake inhibitors. In this context, we previously demonstrated that following chronic intravenous self-administration of cocaine, cocaine upregulated dopamine transporters, while the dopamine-selective uptake inhibitor GBR12909 lacks this effect. New evidence now demonstrates that the self-administration of bupropion, but not nomifensine upregulated dopamine transporters confirming initial results obtained by NIDA IRP scientists. Since the neuroadaptive changes that gradually develop after chronic exposure to cocaine may relate to addictive and withdrawal states, the elucidation of pharmacological heterogeneity among dopamine reuptake inhibitors by examining the differences in the neuroadaptive molecular changes following chronic exposure to these drugs may have direct relevance to the understanding of the neurobiology of cocaine addiction. Tella, S., Ladenheim, B. and Cadet, J.L. Differential Regulation of Dopamine Transporter following Chronic Self-Administration of Bupropion and Nomifensine. J. Pharmacol. Exp. Ther. 281, pp. 508-513, 1997.
Effects of Marijuana on Visual Responses
The results of a clinical study indicate that marijuana impairs smooth pursuit eye tracking and reduces the response to a flash of light. These effects were previously unrecognized. They offer a potentially important explanation for the detrimental effects of marijuana on complex performance tasks and its role in workplace and automobile accidents.
A review of the literature indicates that the reinforcing effects of abused drugs depend on the speed of their delivery to sites in the reward pathway of the brain. Interestingly, therapeutic effects of replacement medications do not depend on the rate of delivery. For example, smoked nicotine is highly addictive but transdermal delivery of nicotine aids in the maintenance of abstinence. A similar situation exists for opiates (smoked or injected heroin versus oral methadone or LAAM). This tenet of drug action may dictate development of medication for the treatment of cocaine and other stimulant abuse.
Acute effects of tobacco withdrawal are diminished by transdermal delivery of nicotine. Although many studies have shown that the nicotine patch and gum increase successful tobacco abstinence this study is unique in that it demonstrates that the patch reduces short-term performance, EEG and cardiovascular measures of tobacco withdrawal. Endocrine and subjective measures of withdrawal were not reduced by the patch. The study also demonstrates the utility of this experimental paradigm for studying tobacco withdrawal.
The acute effects of a proposed treatment for cigarette smoking was evaluated in an outpatient study. Cigarette smoking through an occlusive filter diminishes smoke exposure to men and women. The barrier reduced the plasma nicotine and CO boost ordinarily seen after smoking. Effects on EEG and cardiovascular measures were not significant. Pharmacokinetic
examination indicated that men and women have equal exposure (and health risks) to nicotine after smoking and that kinetic properties of nicotine after smoking are similar to those derived from infusion studies.
Mecamylamine, a centrally acting antagonist of nicotine, caused similar EEG and performance effects in non abstinent smokers and in non smokers. These results indicate that nicotinic cholinergic receptors may modulate EEG and performance. The results further suggest a model to study effects of nicotinic cholinergic deficits (as those seen in Alzheimer's disease) in young healthy individuals. Preliminary evidence indicates that the EEG effects of smokeless tobacco use is similar to that after smoke-delivered nicotine. The stimulatory effects of smokeless tobacco may be related to their appeal and addictive properties. These results are from a controlled laboratory study of smokeless tobacco users.
Preliminary studies with a recently developed denicotinized cigarette indicate that smoked delivered nicotine is responsible for many of the physiologic effects of smoking but the short term subjective effects of smoking may be independent of nicotine delivery.
Hair testing is a new technology that is growing in popularity for use in employment testing and forensic testing for drugs of abuse. Its primary advantage is the long window of detection (months compared to days by urinalysis). There is also interest in use of hair testing in prevalence studies and monitoring drug use during treatment. This technology is controversial because there is little scientific information available on how drugs are deposited in hair. Studies conducted at the IRP revealed that melanin may be the primary site of drug binding. In vitro studies were performed to characterize cocaine binding to dark and light colored ethnic hair types. In vitro binding to hair was selective, reversible and increased linearly with increasing hair concentrations. Scatchard analyses revealed high affinity (6 to 112 nM) and low affinity (906 to 4,433 nM) binding in hair.
Multivariate analysis indicated that significantly greater nonspecific and specific radioligand binding occurred in dark colored hair compared to light hair. Multivariate analysis also demonstrated a significant ethnicity by sex effect on specific and nonspecific binding to hair. These in vitro results suggest there may be significant ethnic bias in hair testing for cocaine.
Saliva testing for drugs of abuse may have advantages over traditional urine drug testing because it can be collected easily and in a non-invasive manner. Because of the short time course of drugs in saliva, the presence of drug in saliva may infer recent drug use. A recent study was made of cocaine and metabolites in saliva following administration by the intravenous, smoked and intranasal routes. The smoked and intranasal routes produced local contamination of saliva as evidenced by elevated saliva/plasma ratios. These ratios normalized within two hours of dosing; the presence of cocaine was detectable for about 12 hours. The duration of pharmacologic effects (increase in pulse and subjective effects) was generally equal to or shorter than detection times of cocaine in plasma and saliva. Overall, the study demonstrated the usefulness of saliva as a test matrix for the detection and measurement of cocaine following administration by different routes of administration.
Development of New Drug Testing Technology
New methods are needed for monitoring patients' drug use during treatment. A recent study at the IRP evaluated skin (stratum corneum) and sebum, for use in monitoring drug use and their possible role in deposition of drugs in hair. Skin was collected by light scraping from the back and sebum was collected by applying 'Sebutape' patches for 1-2 hour intervals to the forehead. Specimens were collected periodically from subjects who received cocaine and codeine administrations. Drug content in skin and sebum was analyzed by gas chromatography/mass spectrometry. Cocaine and codeine were the primary analytes in sebum and stratum corneum. After dosing, these drugs appeared in sebum within 1-2 hours and were detected for 1-2 days. Peak drug concentrations in skin occurred one day after completion of dosing; elimination of drugs continued over the next 1-2 weeks following dosing. Overall, no definitive relationship was observed between drug concentrations in sebum and stratum corneum compared to dose. Interpretation of drug distribution and elimination in sebum and stratum corneum was complicated by possible contamination of specimens with drugs from sweat. The mechanism(s) for deposition of cocaine and codeine in sebum and skin appeared to be complex and could involve transfer of drugs between different body fluids (i.e., sebum and sweat) and other matrices (i.e., skin and hair).
Improved methods for monitoring illicit drug use are needed in clinical treatment trials. A new technique, sweat patch analysis, was used to determine cocaine and opiate use in methadone maintenance patients enrolled in a voucher-based contingency management trial. The results of thrice weekly urine drug tests (EMIT immunoassay cutoffs;300 ng/mL) from 44 human subjects were compared to results of 355 sweat patches that were applied for 7 days. Sweat was analyzed for cocaine and heroin and metabolites by an ELISA immunoassay (cutoffs 10 ng/mL) and confirmed in a subset of samples by GC/MS (cutoffs 5 ng/mL). The accuracy, sensitivity and specificity of sweat ELISA results was high compared to GC/MS analysis (reference standard method). Data from this field trial suggests that once per week sweat testing may be a viable alternate to the three-times-per-week schedule needed for adequate monitoring by traditional urinalysis for cocaine and heroin.
Clinical Pharmacology Branch
Drug Detection and Evaluation
The Drug Evaluation and Classification (DEC) program is used by police agencies to determine if individuals are behaviorally impaired due to drug use, and if impaired, to determine the class of drug(s) causing the impairment. Although widely used, the validity of the DEC evaluation has not been rigorously tested. The primary goal of this study was to determine the validity of the variables of the DEC evaluation in predicting whether research volunteers had been administered ethanol, cocaine, or marijuana; a secondary goal was to determine the accuracy of trained police officers (Drug Recognition Examiner, DRE) in detecting whether subjects had been dosed with ethanol, cocaine, or marijuana. Community volunteers with histories of drug use received ethanol (0, 0.28, 0.52 g/kg), cocaine (4, 48, 96 mg/70 kg), and marijuana (0, 1.75, 3.55% THC) in a double-blind, randomized, within-subjects design. The ability of the DEC evaluation to predict the intake of ethanol, cocaine, or marijuana was optimal when using 17-28 variables from the evaluation. When DREs concluded impairment was due to drugs other than ethanol, their opinions were consistent with toxicology in 44% of cases. These findings suggest that the DEC evaluation can be used to predict accurately acute administration of ethanol, cocaine, or marijuana, and that predictions of drug use may be improved if DREs focused on a subset of variables.
Subjective and Behavioral Effect Profiles of Alcohol and Marijuana
One study compared subjective and behavioral effect profiles of alcohol and smoked marijuana using technology that controlled puffing and inhalation parameters. Male volunteers with histories of moderate alcohol and marijuana use were administered three doses of alcohol (0.25, 0.5, 1.0 g/kg), three doses of marijuana (4, 8, 16 puffs of 3.55% 9-THC), and placebo in random order under double blind conditions in seven separate sessions. BAC (10-90 mg/dl) and THC levels (63-188 ng/ml) indicated that active drug was delivered to subjects dose dependently. Alcohol and marijuana produced dose-related changes on subjective measures of drug effect. Ratings of perceived impairment were identical for the high dose of alcohol and marijuana. Both drugs produced comparable impairment on digit-symbol substitution and word recall tests, but had no effect on time perception and reaction time tests. Alcohol, but not marijuana, slightly impaired performance on a number recognition test. These data are useful for understanding the relative
performance impairment produced by alcohol and marijuana at the delivered doses and the relationship between their subjective and behavioral effects.
In two other studies, researchers investigated the effect of smoked marijuana on four standardized field sobriety tests (FST) that are used to determine whether a person can safely operate a motor vehicle. Subjective effects and D9-tetrahydrocannabinol (THC) plasma concentrations were also measured to correlate with behavioral impairment. In a residential study, 12 volunteers participated in six experimental sessions. At each session, subjects smoked ad lib two half-cigarettes containing 0 or 3.58% THC. Placebo, low, and high doses consisted of two placebo half-cigarettes, one placebo and one active half-cigarette, and two active half-cigarettes, respectively. Subjects received each marijuana dose twice in random order. Marijuana impaired performance on only one FST, the One Leg Stand, by increasing number of hops and times the elevated foot touched the floor to maintain balance. In a nonresidential study, 20 subjects participated in three experimental sessions. At each session, subjects smoked two cigarettes (16 paced puffs) containing 0, 1.75, or 3.55% THC. Marijuana impaired performance on two FST, One Leg Stand and Finger to Nose. The number of times subjects put their foot down and raised their arms to maintain balance and amount of body sway were increased by marijuana in the One Leg Stand test. A dose-dependent increase in number of misses was observed in the Finger to Nose test. In both studies, marijuana produced orderly dose-related increases in subjective ratings of intoxication. THC plasma concentrations peaked immediately after smoking and had declined to 15-28 ng/ml at the time of FST testing (15 min postsmoking). These data suggest a threshold plasma THC level in the 20-25 ng/ml range for marijuana to impair behaviors critical for safe driving.
Research volunteers with histories of drug abuse participated in a single 60-min session. At the start of each session, subjects smoked ad lib one cigarette (own brand) and completed the Questionnaire on Smoking Urges (QSU) and Mood Form (MF). They then listened to six audiotaped scripts that either described a situation in which a person desired a cigarette (urge condition) or contained no mention of cigarettes (no urge). Additionally, each script contained descriptors of positive, negative, or neutral affect. Presentation order of the scripts was counterbalanced across subjects. There was a greater increase in QSU scores after the urge scripts compared to no-urge scripts across all three affect conditions. Urge scripts decreased positive mood and increased negative mood scores across all three affect conditions. In the negative and neutral affect conditions, positive MF scores decreased and negative MF scores increased. In contrast, mood changes were minimal under positive affect conditions. These data suggest that tobacco craving can be experimentally manipulated in a drug-abusing population. This laboratory model of craving will allow further investigation of the factors that modulate tobacco craving.
Clinical Psychopharmacology Section, Clinical Pharmacology Branch
Discovery of Novel Peptidic Dopamine Transporter Ligands By Screening A Positional Scanning Combinatorial Hexapeptide Laboratory
The reinforcing effects of cocaine are thought to result from cocaine binding to the dopamine (DA) transporter, which inhibits DA uptake and increases synaptic DA levels in the mesolimbic system. One strategy for developing medications for treating cocaine addiction is identifying drugs which bind to the DA transporter (DAT ligands) but which do not inhibit DA uptake as effectively as cocaine. Of the numerous known structural classes of DAT ligands, none convincingly inhibit DAT binding without inhibiting DA uptake. The purpose of the present study was to identify members of a novel structural class of DAT ligands and to characterize their interactions at the DA transporter. A positional scanning hexapeptide D-aminoacid library was screened for inhibition of [125I]RTI-55 binding to rat caudate DA transporters. Based on the results, twelve peptides were synthesized. All twelve peptides inhibited [125I]RTI-55 binding to DA transporters with IC50 values, which ranged from 1.8 B5M to 12 B5M. The two most potent peptides were prepared in larger quantities and will be characterized further in various in vitro assays and in vivo using the technique of in vivo microdialysis.
Behavioral Pharmacology Section, Preclinical Pharmacology Laboratory
Motivational Effects of Combining Stimuli Associated with Food and Cocaine Self- Administration
In previous experiments, IRP scientists have shown that combining two discriminative stimuli associated with the same reinforcer increases responding for that reinforcer by 2-3 fold, regardless of whether the reinforcer was food, water, shock-avoidance or cocaine. Compounding two stimuli associated with two different appetitive reinforcers such as food and water also increased responding, although not to the same degree. We have recently shown a similar effect with cocaine self-administration, where combining stimuli associated with cocaine and food reinforcement led to increased responding. These results support the hypothesis that the effects of two stimuli associated with reinforcers from the same incentive class (appetitive) are mutually enhancing. Thus, in some situations a stimulus associated with an alternative reinforcer might increase the motivation to self-administer cocaine. Panlilio, L. V., Weiss, S. J., and Schindler, C. W. Motivational Effects of Compounding Discriminative Stimuli Associated with Food and Cocaine. Psychopharmacology, In press.
Alteration of the Reinforcing and Other Behavioral Actions of Nicotine by Chronic Caffeine Exposure
Epidemiological surveys in humans show that smokers tend to smoke more cigarettes while drinking coffee and they also drink significantly more coffee than nonsmokers. It is now generally accepted that this positive correlation can be ascribed to interactions between nicotine and caffeine -the main psychoactive ingredients of coffee and tobacco, respectively. In previous experiments, we have shown that pretreatment with acute injections of caffeine can selectively increase nicotine self-administration responding in monkeys. Recently we have extended these studies to characterize nicotine-caffeine interactions under conditions of chronic caffeine exposure. Effects of nicotine are examined in rats that are chronically exposed to caffeine added to their drinking water and in monkeys that receive repeated daily i.m. injections of caffeine. Researchers are using several behavioral techniques including i.v. self-administration, drug discrimination, and a fixed-interval schedule of food reinforcement that are commonly used to investigate the behavioral effects of psychostimulants. Results have demonstrated that chronic caffeine exposure appears to (1) markedly enhance acquisition of iv nicotine self-administration in rats and markedly increase rates of responding for nicotine and nicotine intake in monkeys, (2) qualitatively change the discriminative cue of nicotine without altering the rate of acquisition of a nicotine discrimination, but (3) has little impact on the effects of nicotine on schedule-controlled food maintained responding. Thus, findings reveal a rather complex pattern of nicotine-caffeine interactions which appears to depend upon the particular aspect of behavior under examination.
Gasior, M., Shoaib, M., Yasar, S., and Goldberg, S.R. Qualitative Changes in the Discriminative Properties of Nicotine Produced by Chronic Caffeine Exposure in Rats. International Behavioral Neuroscience Society, 6; 54 (P2-30), San Diego, CA, April, 1997.
Jaszyna, M., Gasior, M., Shoaib, M., Yasar, S., and Goldberg, S.R. Effects of Chronic Caffeine Exposure on the Effects of Nicotine on Schedule-Controlled Behavior in Rats. International Behavioral Neuroscience Society, 6:54(P2-32), San Diego, CA, April, 1997.
Yasar, S., Shoaib, M., Gasior, M., Gasior, M., and Goldberg, S.R. Intravenous Nicotine Self-Administration in Squirrel Monkeys: Effects of Timeout Duration and Caffeine Pretreatment. International Behavioral Neuroscience Society, 6:54(P2-35), San Diego, CA, April, 1997.
Yasar, S., Shoaib, M., Gasior, M., Jaszyna, M., and Goldberg, S.R. Facilitation of IV Nicotine Self-Administration in Squirrel Monkeys by Caffeine. Journal of Psychopharmacology, Vol. 11(Suppl)(3):A14, 1997. Presented at the British Association for Psychopharmacology, Cambridge, United Kingdom, July, 1997.
Neuroadaptive Changes Resulting From Chronic Methamphetamine Self-Administration
A series of studies are underway to identify neuroadaptive changes in the dopaminergic (DA transporters, D1 and D2 receptors) and opioid (mu, delta , and kappa receptors) systems occurring at the different stages of withdrawal from chronic exposure to methamphetamine self-administration in animals receiving response-dependent (self-administered) and response-independent (yoked) drug administration. Research is carried out in Sprague-Dawley rats using a variety of experimental procedures, including: intravenous self-administration, quantitative autoradiography and immunocytochemistry. Initial findings confirm previous demonstrations that methamphetamine can serve as a reinforcer of self-administration behavior and support the hypothesis that a functional reduction of dopamine neurotransmission is one important consequence of chronic methamphetamine exposure, unmasked when methamphetamine administration is withdrawn. Findings also indicate a functional reduction in endogenous opioid release, which may be another important component of methamphetamine withdrawal. Stefanski, R., Ladenheim, B., Lee, S.H., Cadet, J.L., and
Goldberg, S.R. Neuroadaptive Changes in Rats After 4 Months of Intravenous Methamphetamine Self-Administration. In L.S. Harris (Ed.). Problems of Drug
Dependence, 1997: Proceeding of the 59th Annual Scientific Meeting, College on Problems of Drug Dependence, NIDA Research Monograph, Washington, D.C., U.S. Gov't Printing Office, In press.
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