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National Institute on Drug Abuse

Director's Report to the National Advisory Council on Drug Abuse
February, 1999

Research Findings

Intramural Research

Behavioral Neuroscience Branch

An Effective Treatment for Cocaine Toxicity

Cocaine abuse is a public health concern as seizures and deaths continue to be reported. There were an estimated 150,000 cocaine-related emergency room incidents in 1995 which accounted for 27% of all emergency-department drug-related episodes. These toxicity data represent an increasing trend since 1990. Current emergency treatments for cocaine overdose are not always effective. IRP investigators now report that dopamine D3 receptor agonists dose-dependently and completely block the convulsant and lethal effects of cocaine. In addition to anticonvulsant effects, these compounds also demonstrated antiepileptogenic properties, blocking the development and expression of kindled seizures (increased sensitivity to cocaine-related toxicities) that result from repeated administration of cocaine. These findings implicate dopamine D3 receptors in the acute and chronic toxicity of cocaine and provide a possible alternative medication for the management of cocaine overdose. Gasior, M. and Witkin, J.M. Cocaine-Kindled Seizures: Some Potential Treatment Modalities. Polish Journal of Pharmacology, 50 (suppl): 39, 1998. Witkin, J.M. and Witkin, J.M. Dopamine D3 Receptor Involvement in the Convulsant and Lethal Effects of Cocaine. Polish Journal of Pharmacology, 50 (suppl), pp. 44-45, 1998.

Neuroactive Steroids: Novel Potential Drug Abuse Treatments

Data are beginning to accumulate to suggest a role for neuroactive steroids in certain phases of drug dependence. Drug withdrawal-related anxiety has been treated with benzodiazepines and related compounds in preclinical models and in clinical practice. IRP investigators have shown that some drugs in this class produce effects in animal models that suggest that they may also be useful in the control of anxiety related to cessation of drug abuse. Specifically, neuroactive steroids both prevented and reversed anxiety-like behavioral effects of pentylenetetrazol, a drug used to model drug withdrawal anxiety. These data in conjunction with safety data on neuroactive steroids thus far reported in humans encourages further investigation into the role of this drug class in drug abuse cessation. Beekman, M., Ungard, J.T., Gasior, M., Carter, R.B., Dijkstra, D., Goldberg, S.R. and Witkin, J.M. Reversal of Behavioral Effects of Pentylenetetrazol by the Neuroactive Steroid Ganaxolone. Journal of Pharmacology and Experimental Therapeutics, 284, pp. 868-877, 1998.

Development and Plasticity Section, Cellular Neurobiology Branch

New Oncogene Developed for Producing Neuronal Cell Lines for Drug Toxicity and Gene Expression Testing

Using cell lines can be of great value for evaluating drug-induced neurotoxicity and changes in gene expression induced by drugs of abuse. Because cell lines are homogeneous, the use of cell lines reduces the complication resulting from the presence of multiple cell types that are present in whole tissue or intact animals. Finding changes in expressed genes can thus be greatly simplified through the use of cell lines. To give an example, drugs of abuse may induce changes in growth factors or adhesion molecules which mediate neuroplastic changes that result in drug adaptation, and thereby contribute to addiction. In addition, the use of human cell lines is the only means of identifying human drug-induced genes, since human subjects cannot be subjected to controlled studies of neurotoxicity. Current methods for generating neural cell lines, however, employ oncogenes which interfere with the development of mature neuronal phenotypic properties, so that model cell lines produced by current cell immortalization techniques are far from ideal for use for in vitro studies of drug effects. The most commonly used oncogene for the generation of neural cell lines is SV40 large T antigen, which has the advantage of effectiveness and well-understood mechanisms of action, but interferes with normal processes related to cell differentiation. A truncated mutant form of SV40 large T antigen, less than one-fourth the size of the intact molecule, and which lacks several of the properties that interfere with normal cellular functions, was developed. This mutant oncogene was able to immortalize mesencephalic cells, and cells produced with this oncogene demonstrated many properties of normal neurons and are sensitive to drug-induced toxicity. This method may be useful for generating neuronal cell lines for examining effects of drugs of abuse in vitro. Truckenmiller, M.E., Tornatore, C., Wright, R.D., Dillon-Carter, O., Meiners, S., Geller, H.M., Freed, W.J. A Truncated SV40 Large T Antigen Lacking the p53 Binding Domain Overcomes p53-induced Growth Arrest and Immortalizes Primary Mesencephalic Cells. Cell and Tissue Research, 291, pp. 175-189, 1998; Dillon-Carter, O., Conejero, C., Tornatore, C., Poltorak, M., and Freed, W.J. N18-RE-105 Cells: Differentiation and Activation of p53 in Response to Glutamate and Adriamycin is Blocked by SV40 Large T Antigen tsA58. Cell and Tissue Research, 291, pp. 191-205, 1998.

Cell Adhesion Molecules in Neuropsychiatric Disorders and Drug Effects

Cell Adhesion molecules are important in the development, maturation, and plasticity of the nervous system. Changes in cell adhesion molecules are also likely to be involved in adaptational changes which occur in response to chronic exposure of the brain to drugs, and may play a role in the establishment of addiction. Neuropsychiatric disorders are also a major risk factor for addictive behavior. We have found changes in two cell adhesion molecules, N-CAM and L1, in the cerebrospinal fluid of patients with schizophrenia, and changes in N-CAM in patients with mood disorders. A novel isoform of N-CAM with a molecular weight of 105-115 kDa has been identified, and is changed in cytosolic fractions of post-mortem brain samples from patients with schizophrenia. A separate isoform of N-CAM, specifically a cytosolic 140 kDa isoform containing the 10 amino acid VASE exon, is changed in the hippocampus of patients with bipolar mood disorder. Interestingly, drug treatment status does not seem to be responsible for these changes; however, similar changes are not seen early in the course of illness. Therefore, it appears that changes in cell adhesion molecules may be linked to long-term chronic drug exposure. The fact that drug treatment produces long-term changes in cell adhesion molecules may be significant in terms of adaptations that occur following chronic exposure to drugs. Such changes, or similar changes, may be important in the establishment or maintenance of addiction. Vawter, M.P., Hemperly, J.J., Hyde, T.M., Bachus, S.E., VanderPutten, D.M., Howard, A.L., Cannon-Spoor, H.E., McCoy, M.T., Webster, M.J., Kleinman, J.E., Freed, W.J. VASE-Containing N-CAM Isoforms are Increased in the Hippocampus in Bipolar Disorder but not Schizophrenia. Experimental Neurology, 154, pp. 1-11, 1998. Vawter, M.P., Cannon-Spoor, H.E., Hemperly, J.J., VanderPutten, D.M., Hyde, T.M., Kleinman, J.E., Freed, W.J. Abnormal Expression of Cell Adhesion Molecules in Schizophrenia. Experimental Neurology, 149, pp. 424-432, 1998. Van Kammen, D.P,. Poltorak, M. Kelley, M.E., Yao, J.K., Gurklis, J.A., Peters, J.L., Hemperly, J.J., Wright, R.D., Freed, W.J. Further Studies of Elevated Cerebrospinal Fluid Neural Cell Adhesion Molecule in Schizophrenia. Biological Psychiatry, 43, pp. 680-686, 1998.

Cellular Neurophysiology Section, Cellular Neurobiology Branch

Transplantation of Fetal Kidney Tissue Reduces Infarction Volume of Cerebral Cortex in Adult Rats

It has been shown that neurotrophic factors have both regenerative and protective actions on neurons in vivo and in vitro. IRP investigators and others recently reported that intracerebral administration of glial cell line derived neurotrophic factor (GDNF) protects against ischemia-induced injury in the cerebral cortex of adult rats. Since fetal kidney tissues contain a number of trophic factors such as GDNF, neurturin, transforming growth factor- b, and OP-1, it is possible that transplantation of fetal kidney tissue could protect brain against ischemia-induced injury by providing these trophic factors. Kidneys from rat embryos, at gestational day of 16, or from adult rats, were dissected and cut into small pieces. Adult male Sprague-Dawley rats were anesthetized with chloral hydrate and placed in a stereotactic apparatus. Kidney tissues were transplanted into 3 cortical areas adjacent to the right middle cerebral artery. Thirty minutes after transplantation, the right MCA was ligated with a 10-O suture and both common carotid arteries were occluded with non-traumatic arterial clips for 90 min. Twenty-four hours after reperfusion, animals were anesthetized with urethane and perfused intracardially with saline. Brain tissue was removed, sliced, incubated with 2% triphenyltetrazolium chloride for 30 min, and then transferred into 5% formaldehyde solution for fixation. The volume of cortical infarction was measured in each slice and summed using computerized planimetry. Findings indicate that transplantation of either adult kidney tissue or injection of vehicle did not reduce the infarction volume of the cerebral cortex. On the other hand, animals which received fetal kidney transplant showed a significant reduction of infarction volume. Taken together, these data show that fetal kidney transplantation reduces ischemia-induced injury in the cerebral cortex. Authors suggest a release of trophic factors may be involved since adult kidney tissues contain much smaller amounts of these factors. Chiang, Y. H., Chiou, A.L., Lin, S.Z., Hoffer, B. J., and Wang, Y. Transplantation of Fetal Kidney Tissue Reduces Infarction Volume of Cerebral Cortex in Adult Rats. Soc. Neurosci. meeting, 1998.

Co-administration of 1,25(OH)2 Vitamin D3 and Retinoic Acid Attenuates Cortical Infarction Induced by Middle Cerebral Arterial Ligation

IRP investigators have previously reported that intracerebral administration of glial cell line derived neurotrophic factor diminishes middle cerebral arterial (MCA)-ligation-induced cortical infarctions in rats. Recent studies have indicated that application of 1,25(OH)2 Vitamin D3 [1,25(OH)2D3] with retinoic acid enhances GDNF mRNA expression in vitro. The purpose of this study is to investigate if co-administration of 1,25(OH)2D3 and retinoic acid protects against ischemic brain injury. Adult male Sprague-Dawley rats were chronically injected with 1,25(OH)2D3 for 3 days. Animals were later anesthetized with chloral hydrate. Low dosage of retinoic acid was given intraventricularly and directly to the cortex, adjacent to the MCA. Right MCA was temporarily ligated with 10-O suture for 90 minutes. Animals were sacrificed for TTC staining after 24 hour reperfusion. Investigators found that pretreatment of 1,25(OH)2D3 or retinoic acid separately did not attenuate the injury. Co-administration of these two compounds, however, greatly reduced brain infarction. In conclusion, these data suggest that co-administration of 1,25(OH)2D3 and retinoic acid prevents against ischemia-induced brain damage, possibly mediated through the activation of neurotrophic factors. Wang, Y., Lin, S.Z., Chiang, Y. S., and Hoffer, B.J. Co-administration of 1,25(OH)2 Vitamin D3 and Retinoic Acid Attenuates Cortical Infarction Induced by Middle Cerebral Arterial Ligation. Soc. Neuroscience meeting, 1998.

GDNF Protects Against Ischemia-induced Injury in the Cerebral Cortex

Glial cell line-derived neurotrophic factor (GDNF), a recently described and cloned member of the TGF- b superfamily, has been shown to have marked trophic activity on several populations of central neurons. Survival-promoting and injury protectant activity in vitro and in vivo, using several paradigms, has been demonstrated for ventral mesencephalic dopaminergic neurons and spinal cord motoneurons. In view of a proposed commonality of mechanisms, involving intracellular free radical generation, depolarization-induced Ca++ influx, and mitochondrial respiratory enzyme injury, between such GDNF-responsive paradigms and those of ischemia-induced injury, IRP scientists tested the effects of GDNF on the extent of neural degeneration induced by transient middle cerebral artery (MCA) occlusion. Authors now report that intracerebroventricular and intraparenchymal administration of GDNF potently protects the cerebral hemispheres from damage induced by MCA occlusion. In addition, the increase in nitric oxide which accompanies MCA occlusion and subsequent reperfusion is almost completely blocked by GDNF. Thus, this peptide may play an important role in the treatment of cerebrovascular occlusive disease. Wang, Y., Lin, S.Z., Chiou, A. L., and Hoffer, B.J. GDNF Protects Against Ichemia-Induced Injury in the Cerebral Cortex. J. Neurosci., 17, pp. 4341-4348, 1997.

Molecular Neuropsychiatry Section, Cellular Neurobiology Branch

The Role of Reactive Oxygen Species in METH-induced Toxicity

In order to test the idea of the involvement of superoxide radicals in METH-induced toxicity, IRP scientists assessed the effects of toxic doses of the drug on antioxidant defense systems in the brains of mice. Because METH- induced toxicity is attenuated in copper/zinc-superoxide dismutase transgenic (Cu/Zn-SOD-Tg) mice, investigators sought to determine if METH had differential effects on antioxidant enzymes on these mice in comparison to non-Tg mice. A toxic dose of METH caused a significant decrease in Cu/Zn-SOD activity in the cortical region without altering enzyme activity in the striata of non-Tg mice. CuZn SOD activity was not affected in the brains of heterozygous SOD-Tg mice, whereas there was a small increase in the striata of homozygous SOD-Tg mice. In addition, METH caused decreases in catalase (CAT) activity in the striatum of non-Tg mice and significant increases in the cortex of homozygous SOD-Tg mice. METH also induced decreases in glutathione peroxidase (GSH-Px) in both cortical and striatal regions of non-Tg mice and in the striatum of heterozygous SOD-Tg mice, whereas, this enzyme was not affected in the homozygous SOD-Tg mice. Interestingly, lipid peroxidation was markedly increased in both cortices and striata of non-Tg and heterozygous SOD-Tg mice, but not in the homozygous SOD-Tg. When taken together, these results suggest that the toxic effects of METH involve not only superoxide radicals but a cascade that also includes hydrogen peroxide and hydroxyl radicals. In vitro studies in the laboratory have now documented the production of both superoxide radicals and hydrogen peroxide during exposure of cells to METH. Investigators from other laboratories have reported that hydroxyl radicals are also involved in METH-induced toxicity. Subramaniam J., Ladenheim B, and Cadet J.L. Methamphetamine- induced Changes in Antioxidant Enzymes and Lipid Peroxidation in Copper/Zinc Superoxide Dismutase Transgenic Mice. Ann. NY Acad. Sci., 844, pp. 92-102, 1998.

Superoxide Radicals are Mediators of the Effects of METH on Zif268 (Egr-1, NGFI-A) in the Brain

The cellular effects of exogenous and endogenous ligands are mediated via a molecular cascade that involves the activation of a number of transcription factors. These include the fos and jun families as well as Zif268 (also known as Egr-1, Krox24, or NGFI-A) that have been shown to be rapidly activated by several signaling agents. More recently, the role of dopaminergic agents such as abusable stimulants as activators of these IEGs have begun to be actively studied because of the possibility that they might provide insights into molecular events that lead to stimulant-induced behavioral sensitization in animals. Recent studies aimed at detailing the molecular events involved in the action of stimulant drugs have documented the fact that the amphetamines can activate IEGs such as c-Fos and Zif268. These increases occur very rapidly after drug administration and return to baseline within a few hours. Because of the robustness and consistency of these responses, it has been suggested that IEGs might be important mediators of the neuroplastic adaptation which occurs in the brain during chronic administration of these stimulants. This activation of IEGs cause by these drugs is thought to be mediated mainly through stimulation of D1 receptors. IRP scientists reasoned, however, that redox status might also affect Zif268 expression. As a first step towards assessing a possible role for free radicals in METH-induced changes in IEGs, these investigators used CuZn superoxide dismutase (SOD) transgenic (Tg) mice and quantified the effects of METH on c-Fos and Zif268 mRNAs by in situ hybridization techniques. Mice were injected with 25 mg/kg of METH and sacrificed at various time points afterwards. There were significant METH-induced increases in both c-Fos and Zif268 mRNAs in the frontal cortex and striatum of both strains of animals. Interestingly, the increases in Zif268 were markedly attenuated in the CuZn SOD-Tg mice; the increases in c-Fos were also attenuated, but to a significantly lesser degree. These results indicate that superoxide radicals might play an important role in the activation of Zif268 after METH administration. When taken together with similar findings for AP-1, these results also raise the possibility that oxidative mechanisms might be important factors in neuroadaptive molecular changes caused by stimulant drugs. Hirata H., Asanuma M., and Cadet J.L. Superoxide Radicals are Mediators of the Effects of Methamphetamine on ZIF 268 (Egr-1, NGFI-A) in the Brain: Evidence from Using CuZn Superoxide Dismutase Transgenic Mice. Mol. Brain Research, 58, pp. 209-216, 1998.

Melatonin Attenuates Methamphetamine-induced Toxic Effects on Dopamine and Serotonin Terminals in Mouse Brain

Methamphetamine (METH) is a drug of abuse that causes deleterious effects to brain monoaminergic systems. These toxic effects are thought to be due to oxidative stress. The pineal hormone, melatonin, has been shown to have neuroprotective effects against toxic quinones and oxidative stress produced by catecholamines. The present study was thus undertaken to assess possible protective effects of melatonin against METH-induced neurotoxic effects on the striatum and the nucleus accumbens by using autoradiographic techniques. Four dosages (5, 20, 40, 80 mg/kg) of melatonin were administered to mice intraperitoneally 30 minutes prior to the injections of METH (4 x 5 mg/kg) given at 2- hour intervals. The lowest doses of melatonin (5 mg/kg) had no significant effects against METH-induced toxicity. However, the higher doses (40 or 80 mg/kg) of melatonin significantly attenuated METH- induced toxic effects on both dopamine and serotonin systems. These data provide further evidence for a possible role of oxidative stress in METH-induced toxicity. Hirata H., Asanuma M., and Cadet J.L. Melatonin Attenuates Methamphetamine-induced Toxic Effects on Dopamine and Serotonin Terminals in Mouse Brain. Synapse, 30, pp. 150-155, 1998.

Toxicity of 6-OHDA is Attenuated in SOD Mice

6-Hydroxydopamine is a neurotoxin that produces degeneration of the nigrostriatal dopaminergic pathway in rodents. Its toxicity is thought to involve the generation of superoxide anion secondary to its autoxidation. To examine the effects of the overexpression of CuZn SOD activity on 6-hydroxydopamine- induced dopaminergic neuronal damage, IRP scientists have measured the effects of 6-hydroxydopamine- on striatal and nigral dopamine transporters and nigral tyrosine hydroxylase-immunoreactive neurons in CuZn SOD Tg mice. Intracerebroventricular injection of 6-hydroxydopamine (50 mg) in non-transgenic mice produced reductions in the size of striatal area and an enlargement of the cerebral ventricle on both sides of the brains of mice sacrificed two weeks after the injection. In addition, 6-hydroxydopamine caused marked decreases in striatal and nigral [125I]RTI-121-labelled dopamine transporters not only on the injected side but also on the non-injected side of non-transgenic mice; this was associated with decreased cell number and size of tyrosine hydroxylase-immunoreactive dopamine neurons in the substantia nigra pars compacta on both sides in these mice. In contrast, SOD Tg mice were protected against these neurotoxic effects of 6-OHDA, with the homozygous transgenic mice showing almost complete protection. These results provide further support for a role of superoxide anion in the toxic effects of 6-OHDA. They also provide further evidence that reactive oxygen species may be the main determining factors in the neurodegenerative effects of catecholamines. These studies document important similarities between the mechanisms involved in catecholamine-induced and METH-induced toxicity. Asanuma M., Hirata H., and Cadet J.L. Attenuation of 6-hydroxydopamine-induced Dopaminergic Nigrostriatal Lesions in Superoxide Dismutase Transgenic Mice. Neuroscience, 85, pp. 907-917, 1998.

Endogenous Opioid Peptide as a Free Radical Scavenger: Prevention of the Neurotoxicity Induced by Methamphetamine ("Ice")

DADLE is a stable analog of the endogenous delta opioid peptide Leu-enkephalin. Over the past few years, IRP researchers in collaboration with scientists from University of Kentucky and University of Michigan have demonstrated the remarkable tissue protective properties of DADLE in extending the survival of isolated peripheral organs. DADLE was found to dramatically enhance the survival of isolated lungs, and isolated hearts, both of which are known to be difficult to preserve. As the survival of tissue depends largely on the oxidative state of the tissue which is linked intimately with the formation of free radicals, the researchers reason that perhaps one of the mechanisms of DADLE in enhancing the tissue survival is to act as a free radical scavenger. As such, it is not unreasonable to speculate that DADLE may act as a tissue protective agent even in the central nervous system. The IRP researchers, in collaboration with intramural scientists from NIMH, demonstrated that indeed DADLE is a free radical scavenger sequestering two of the most reactive free radicals: superoxide anion and hydroxyl radical. Further, given before methamphetamine, DADLE was shown to block the long-term damage of dopaminergic nerve terminals induced by methamphetamine. These data provide a new avenue for understanding the pathobiological damage caused by psychostimulants and the potential treatment strategies thereof. As methamphetamine-induced neurotoxicity is believed to be a potential model system for Parkinsonism, these results suggest that perhaps the endogenous opioid peptides play an important role in the pathogenesis of Parkinsonism. Tsao, L.-I., Ladenheim, B., Andrews, A., Chiueh, C.C., Cadet, J.L. and Su, T.-P. Delta opioid peptide [D-Ala2,D-Leu5] Enkephalin Blocks the Long-Term Loss of Dopamine Transporter Induced by Multiple Administrations of Methamphetamine: Involvement of Opioid Receptors and Reactive Oxygen Species. J. Pharmacol. Exp. Ther., 287, pp. 322-331, 1998.

Sigma Receptor Agonists Potentiate the Action of an Antipsychotic Agent Sulpiride in Primary Cortical Neurons

Sigma receptor agonists have been shown by IRP researchers to play a beneficial role in several animal models of amnesia, including those related to aging and Alzheimer's disease. The underlying mechanism of action of those sigma agonists have been speculated to be related to their ability to potentiate the action of endogenous glutamate on the NMDA receptors which is known to be involved in learning and memory. One of the mechanisms of action of antipsychotic agent sulpiride has been speculated to be related to the NMDA receptors by creating a "hyper-NMDA" state which would benefit the schizophrenic in theory. IRP researchers, in collaboration with Japanese scientists, found that sulpiride can potentiate the NMDA-induced intracellular Ca2+ concentration ([Ca2+]i). Also, they found that sigma receptor agonists, by their own having a tendency to potentiate the action of NMDA, could further enhance the NMDA-potentiating action of sulpiride. Furthermore, they found that this action of sulpiride is mediated by a protein-kinase related intracellular action. These data indicate that antipsychotic sulpiride exerts its therapeutic efficacy via an intra- cellular biochemical action and that sigma agonists, in addition to being antiamnesic, may be beneficial adjunct therapeutic agents for treating schizophrenia. Hayashi, T., Su, T.-P., Kagaya, A., Nishida, A., Shimizu, M. and Yamawaki, S. Neuroleptics with Differential Affinities at Dopamine D2 Receptors and Sigma Receptors Affect Differently the N-methyl- D-aspartate-induced Increase in Intracellular Ca2+ Concentration in Rat Frontal Cortical Neurons: Involvement of Protein Kinase. Synapse, 31, pp. 20-28, 1999.

Chemistry and Drug Metabolism Section, Clinical Pharmacology Branch

Differentiating New Marijuana Use from Residual Drug Excretion in Occasional Marijuana Users

A drug dose is generally excreted in decreasing amounts in sequential urine specimens for only a few days following drug administration. The concentration of the drug in urine may vary considerably due to the amount of urine in the bladder; increases in urine drug concentration may be mistakenly interpreted as new drug use rather than residual drug excretion. Normalization of drug excretion to the urine creatinine concentration reduces the variability of drug measurement due to urine dilution and is suggested for use in treatment and employee assistance programs. A controlled clinical study was recently completed to provide data for the interpretation of urine cannabinoid test results. These data indicate selection of a ratio threshold to evaluate sequential creatinine normalized urine drug concentrations can improve the ability to distinguish residual excretion from new drug usage. The selection of an appropriate ratio can be made based on the needs of a specific urine drug testing program taking into account sensitivity, specificity, and accuracy data. Huestis, M.A., and Cone, E. Urinary Excretion Half-life of 11-Nor-9-carboxy-delta 9-tetrahydrocannabinol in Humans. Therapeutic Drug Monitoring, 20, pp. 570-576, 1998; Huestis, M.A., and Cone, E. Differentiating New Marijuana Use From Residual Drug Excretion in Occasional Marijuana Users. Journal of Analytical Toxicology, 22, pp. 445-454, 1998. Cone, E.J., Lange, R., and Darwin, W.D. In Vivo Adulteration: Excess Fluid Ingestion Causes False Negative Marijuana and Cocaine Urine Test Results. Journal of Analytical Toxicology, 22, pp. 460-473, 1998.

Saliva and Plasma Testing for Drugs of Abuse: Comparison of the Disposition and Pharmacological Effects of Cocaine

The pharmacological effects and disposition of cocaine in plasma and saliva following subcutaneous drug administration were evaluated. Pharmacological effect data (pupil diameter, heart rate, blood pressure, subject-reported "High") were compared to concurrent cocaine and metabolite concentrations determined by GC-MS. Cocaine was detected in both saliva and plasma within 5-10 min following dosing, and cocaine concentrations in both matrices peaked within 30-60 min. Saliva/plasma cocaine ratios were generally greater than 2 in specimens collected for up to 24 hr following dosing. Pharmacological effects observed after cocaine administration included increases in heart rate, elevations in blood pressure, dilation of pupils, and increases in subject-reported "High". The duration of pharmacological effects was consistently shorter than, or similar to the time course for detection of cocaine in saliva and plasma. These findings suggest that saliva testing may provide valuable insights into the onset and duration of cocaine-induced pharmacological effects and appears to be a useful alternative to plasma testing for cocaine. Cone, E.J., Tsadik, A., Oyler, J. and Darwin, W.D. Cocaine Metabolism and Urinary Excretion Following Different Routes of Administration. Therapeutic Drug Monitoring, 20, pp. 556-560, 1998.

Clinical Psychopharmacology Section, Clinical Pharmacology Branch

GBR12909 Decanoate Affords Long-term Antagonism of Methamphetamine-induced Dopamine Release

Methamphetamine (METH) addiction is a growing health concern worldwide, yet no medicines for this disease are available. Recent findings indicate that DL699, a decanoate analog of GBR12909, can decrease METH self-administration in animals. In the present work, IRP investigators used in vivo microdialysis to evaluate the effect of DL699 pretreatment on METH-induced elevations in extracellular dopamine (DA) in rat nucleus accumbens. Rats received a single im injection of DL699 or its oil vehicle on Day 1; rats were subsequently tested on Days 6 and 13. Microdialysis probes were inserted into previously implanted guide cannulae and perfused with Ringers= solution overnight. In the morning, dialysate samples were collected every 20 min and assayed for DA by HPLC-EC. Basal dialysate DA levels were significantly elevated (2-fold) in the DL699 group on both test days. Moreover, DL699-pretreated rats displayed dramatic and persistent reductions in METH-induced stimulation of DA release when compared to vehicle-pretreated rats. Autoradiographic assessments of [125I]-RTI-55 binding showed the density of DA transporter sites was diminished in the DL699 group. These data suggest that DL699 can antagonize the actions of METH on mesolimbic DA neurons and this effect involves blockade of DA transporters. Thus, DL699 or similar agents might be useful medications for treating METH addiction. Baumann, M.H., Ayestas, M.A., Lewis, D.B., Rice, K.C., and Rothman, R.B. GBR12909 Decanoate Affords Long-term Antagonism of Methamphetamine-induced Dopamine Release. 28th Annual Meeting, Society for Neuroscience, Los Angeles, CA, November, 1998.

Identification of a Novel Cocaine Binding Site in Brain Membranes Prepared from Dopamine Transporter Knockout Mice

Previous work has suggested that the cocaine analog [125I]RTI-55 labels a novel binding site in rat brain membranes, termed DATsite2, which is not associated with the dopamine (DA), serotonin (5-HT) or norepinephrine (NE) transporters (JPET, 274, pp. 385-395, 1995). In this study IRP investigators tested whether DATsite2 is a product of the DA transporter (DAT) gene. A T-antigen knockin at the DAT gene that results in an effective DAT KO, originally developed at NIDA's IRP and now bred at the GRC, were used. Lack of the DAT gene was confirmed by southern blots. Brain membranes were prepared from frozen whole brain minus caudate of swiss-webster (SW) mice, +/+ mice, +/- mice and -/- mice. KO mice were used at approximately 23 days old. Binding surface analysis of [125I]RTI-55 binding to SW membranes, with 100 nM citalopram to block binding to the 5-HT transporter (SERT), revealed two binding sites: DAT (Bmax = 0.63, Kd = 1.8 nM, Ki of RTI-113 = 18 nM) and DATsite2 (Bmax = 1.48, Kd = 28.3 nM, Ki of RTI-113 = 2299 nM), replicating studies conducted with rat brains. [125I]RTI-55 binding blocked with 100 nM citalopram (DAT binding condition) was reduced by 82% in the -/- mice compared to the +/+ mice. [125I]RTI-55 binding in the presence of 100 nM GBR12935 (SERT binding condition) was reduced by 40% in the -/- mice compared to the +/+ mice. With SERT blocked by 100 nM citalopram, 100 nM RTI-113 inhibited [125I]RTI-55 binding by 64% in +/+ mice, 49% in +/- mice and 17% in -/- mice, consistent with an elimination of DAT in the -/- mice. In contrast, the inhibition produced by benztropine (400 nM), which has equal affinity for DAT and DATsite2, was similar in +/+, +/- and -/- mice. Viewed collectively, these data support the hypothesis that DATsite2 is not a product of the DAT gene. The DAT knockout mouse will be a useful system for characterizing DATsite2. Rothman, R.B., Dersch, C.M., Uhl, G.R., Carroll, F.I., Rowley, D.L., and Donovan, D. Identification of a Novel Cocaine Binding Site in Brain Membranes Prepared from Dopamine Transporter Knockout Mice. 28th Annual Meeting, Society for Neuroscience, Los Angeles, CA, November, 1998.

Alterations in Serotonergic Responsiveness during Cocaine Withdrawal in Rats: Similarities to Major Depression in Humans

Withdrawal from long-term cocaine use is accompanied by symptoms resembling major depression. Because acute cocaine affects serotonin (5-HT) neurons, and 5-HT dysfunction is implicated in the pathophysiology of depression, we evaluated the effects to 5-HT agonists in rats withdrawn from repeated injections of cocaine (15 mg/kg i.p., b.i.d., 7 days) or saline. In the first study, prolactin (PRL) responses elicited by the 5-HT-releasing agent fenfluramine, the 5-HT1A agonist (+/-)-8-hydroxy-2-(di-n- propylamino)tetralin (8-OH-DPAT), and the 5-HT2A/2C agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) were examined as indices of postsynaptic 5-HT receptor function. In a second study, specific responses induced by 8-OH-DPAT, namely inhibition of brain 5-HT synthesis and stimulation of feeding, were examined as correlates of 5-HT1A autoreceptor function. Prior treatment with cocaine did not modify fenfluramine-evoked PRL release; however, the PRL secretory response to 8-OH-DPAT was blunted and the PRL response to DOI was potentiated after chronic cocaine treatment. Cocaine exposure did not alter the inhibitory effect of 8-OH-DPAT on 5-HT synthesis. 8-OH-DPAT-induced feeding was influenced by prior cocaine, but this effect was secondary to pronounced baseline hyperphagia in the cocaine-treated group. These data indicate that withdrawal from chronic cocaine renders specific subpopulations of postsynaptic 5-HT1A receptors subsensitive and 5-HT2A/2C receptors supersensitive. No evidence for cocaine-induced changes in 5-HT1A autoreceptor responsiveness was found. A survey of the literature reveals similarities in the profile of 5-HT dysfunction between rats withdrawn from cocaine and humans diagnosed with depression. Authors propose that withdrawal from chronic cocaine in rats may serve as a useful animal model of depressive disorders. Baumann, M.H., and Rothman, R.B. Alterations in Serotonergic Responsiveness during Cocaine Withdrawal in Rats: Similarities to Major Depression in Humans. Biol. Psychiatry, 44, pp. 578-591, 1998.

Brain Imaging Section, Neuroimaging Branch

New Compounds with High Affinity for Nicotinic Acetylcholine Receptors Identified as Promising Radioligand Imaging Candidates

3-(2(S)-Azetidinylmethoxy)pyridine (A-85380) has been identified recently as a ligand with high affinity for nicotinic acetylcholine receptors (nAChRs). IRP investigators synthesized a series of ten pyridine-modified analogs of A-85380, featuring a halogen substituent at position 2, 5, or 6 of the 3-pyridyl fragment, and studied their binding to nAChRs in vitro. Authors found that the novel compounds substituted at position 5 or 6, as well as the 2-fluoro derivative, possessed subnanomolar affinity for nAChRs in membranes from rat brain. For these ligands, Ki values ranged from 11 to 210 pM, as measured by competition with (_)-[3H]epibatidine. In contrast, 2-chloro, 2-bromo, and 2-iodo analogs exhibited substantially lower affinities that are likely attributable to their bulkiness at position 2 producing notable changes in the molecular geometry. The high affinity members of the series and (+)-epibatidine displayed a tight fit superposition of low-energy stable conformers. These new high affinity compounds for nAChRs are promising candidates for development as radioligands to study nAChRs both in vitro and in vivo. Koren, A.O., Horti, A.G., Mukhin, A.G., Gundisch, D., Kimes, A.S., Dannals, R.F., and London, E.D. 2-, 5- and 6-Halo-3-(2(S)-azetidinylmethoxy)pyridines: Synthesis, Affinity for Nicotinic Acetylcholine Receptors, and Molecular Modeling. J. Med. Chem. 41, pp. 3690-3698, 1998.

Nicotinic Receptors in the Monkey Brain Successfully Imaged using SPECT -using 5--[123I]iodo-A-85380

The distribution and kinetics of 5-[123I]iodo-A-85380, a novel ligand for brain nicotinic acetylcholine receptors (nAChRs), have been evaluated in the Rhesus monkey using single photon emission computed tomography (SPECT). Peak brain levels of radioactivity were measured in brain at 90 min after injection of the tracer, and radioactivity levels persisted for at least 4 hr. Accumulation of radioactivity was highest in the thalamus, intermediate in frontal cortex and basal ganglia, and lowest in the cerebellum. In other experiments, specific binding was effectively reduced by the subcutaneous injection 1 mg/kg of the nicotinic agonist cytisine 2.25 hr. after radiotracer administration. At 2.5 hr. after cytisine administration, radioactivity in the thalamus was reduced by 84%, in the frontal cortex by 76%, and in the basal ganglia by 57% of the level measured at the time of cytisine administration thereby demonstrating the reversible nature of the binding. On the basis of these findings, together with other data indicating high affinity, receptor subtype selectivity, low non-specific binding, and lack of toxicity in animals, 5-[123I]iodo-A-85380 appears to be a promising ligand for SPECT imaging of nAChRs in the human brain. Chefer, S.I., Horti, A.G., Lee, K.D., Koren, A.O., Jones, D.W., Gorey, J., Links, J.M., Mukhin, A.G., Weinberger, D.R., and London, E.D. In Vivo Imaging of Brain Nicotinic Receptors with 5-[123I]iodo-A-85380 Using Single Photon Emission Computed Tomography. Life Sci. 63, (25), PL355-PL360, 1998.

A Simple Probe Device has Proven Useful in External Imaging of Cholinergic Activity in Brain

Using radiotracers that bind specifically to receptors for drugs and neurotransmitters, it is possible to monitor the time-course of drug action in brain. Specifically, one can monitor the occupancy of brain receptors by drugs that interact directly with such receptors by determining competition of those drugs with the relevant radiolabeled ligands. In addition, if a drug affects the level of an endogenous neurotransmitter, the time-course of this effect might be charted as the competition of the transmitter with the radioligand. This principle has been demonstrated using [125I]dexetimide, a ligand for muscarinic acetylcholine receptors, and a simple gamma probe to monitor the time-course of increase in brain acetylcholine levels externally in mouse brain following the peripheral administration of physostigmine. The use of such a probe system rather than ex vivo assay allows repeated determinations. This relatively inexpensive and simple technology should be considered for external imaging when anatomical resolution is not essential or when more complicated procedures (e.g., PET, SPECT scanning are not available). Sanchez-Roa, P.M., Wagner Jr., H.W., Villemagne, V.L., London, E.D., and Lever, J.R. Effects of Extracellular Acetylcholine on Muscarinic Receptor Binding Assessed by [125I]dexetimide and a Simple Probe. Eur. J. Pharmacol., 358(3), pp. 207-211, 1998.

Injection of Methylnaloxonium into the Locus Coeruleus of Opiate-dependent Rats Elicits Widespread Changes in Brain Glucose Metabolism Similar to Those of Systemically Administered Naloxone

Previous studies have demonstrated a widespread stimulation of regional cerebral metabolic rate(s) for glucose (rCMRglc) in morphine-dependent rats undergoing precipitated opioid withdrawal following the systemic injection of naloxone. Nonetheless, many of the behavioral signs of opioid withdrawal are produced by intracerebral injections of the opioid antagonist, methylnaloxonium (MN), into the locus coeruleus (LC). The present work determined the extent to which cerebral metabolic alterations in opioid withdrawal could be initiated by a local action in LC. Intracerebral injections of MN into LC increased rCMRglc in morphine-dependent rats, and the anatomical distribution of this effect was similar to that produced by systemic injections of naloxone. These data support the view that LC is a major substrate of opioid withdrawal in the brain, and that the change rCMRglc during precipitated opioid withdrawal is largely due to the effects of antagonists in the LC. Kimes, A.S., Maldonado, R, Ambrosio, E., Koob, G.F., and London E.D. Cerebral Glucose Metabolism during Opioid Withdrawal following Methylnaloxonium Injection into the Locus Coeruleus. Brain Res., 814, pp. 1-12, 1998.

2-[18F]fluoro-A-85380 holds Promise as a Useful Radiotracer for Imaging of Brain Nicotinic Acetylcholine Receptors with PET

The in vivo brain regional distribution of 2-[18F]fluoro-A-85380, a novel tracer for positron emission tomographic (PET), followed the regional densities of brain nicotinic acetylcholine receptors (nAChRs) reported in the literature. Evidence of binding to nAChRs and high specificity of the binding in vivo was demonstrated by inhibition with nAChR selective ligands as well as with unlabeled 2-fluoro-A-85380. A preliminary study of 2-fluoro-A-85380 demonstrated a relatively low incidence of acute biological effects indicative of toxicity. Horti, A.G., Scheffel, U., Koren, A.O., Ravert, H.T., Mathews, W.B., Musachio, J.L., Finley, P.A., London, E.D., and Dannals, R.F. 2-[18F]Fluoro-A-85380, An In Vivo Tracer for the Nicotinic Acetylcholine Receptors. J. Nucl. Med. Biol., 25, pp. 599-603, 1998.

High Affinity N-[11C]methylated Analogs of Epibatidine Developed as PET Radiotracers for Nicotinic Acetylcholine Receptors

Four halogen-substituted analogs of N-methyl-epibatidine, a nicotinic acetylcholine receptor (nAChR) ligand, were synthesized. They were [(_)-exo-N-methyl-2-(2-halogeno-5-pyridyl)-7-azabicyclo[2.2.1]heptanes where halogeno = F (1a), Cl (2a), Br (3a), I (4a)]. (_)-N-Ethyl epibatidine (2b) also was synthesized. The compounds (1a, 2a, 3a, and 4a) and their corresponding normethyl analogs (1, 2, 3, and 4) inhibited the in vitro binding of [3H]epibatidine to nAChRs to a similar degree, with affinities in the 27 - 50 pM range. The binding affinity of N-ethylepibatidine (2b), however, was substantially lower. The N-[11C]methyl derivatives of 1, 2, and 3 were synthesized from high specific radioactivity [11C]methyl iodide using a high temperature/high pressure technique. The pattern of regional distribution of compounds [11C]1a, [11C]2a, and [11C]3a in the mouse brain following intravenous administration matched those of three highly specific nAChR probes, [3H]epibatidine, [3H]norchloroepibatidine and (_)-exo-2-(2-[18F] fluoro-5- pyridyl)- 7-azabicyclo-[2.2.1]heptane ([18F]FPH) demonstrating these high affinity radioligands can be used with PET (positron emission tomography) to image nAChRs. Horti, A.G., Scheffel, U., Kimes, A.S., Musachio, J.L., Ravert, H.T., Mathews, W.B., Zhan, Y., Finley, P.A., London, E.D., and Dannals R.F. Synthesis and Evaluation of N-[11C] Methylated Analogs of Epibatidine as Tracers for Positron Emission Tomographic Studies of Nicotinic Acetylcholine Receptors. J. Med. Chem., 41(22), pp. 4199-4206, 1998.

Potent Sigma-1 Receptor Ligand Ameliorates Neuronal Ischemic Injury -- Mechanism Involves Modulation of NMDA-evoked Nitric Oxide Production

Previous work by investigators at NIDA's Intramural Research Program and the Johns Hopkins school of Medicine has shown that 4-phenyl-1-(4-phenylbutyl)piperidine (PPBP), a sigma-1 receptor ligand, ameliorates ischemic neuronal injury measured in in vivo and in vitro models. Recently published work demonstrated that PPBP decreased infarction size, but that prolonged treatment with this compound is associated with loss of therapeutic efficacy. Because NMDA-evoked synthesis of nitric oxide (NO) may play an important role in excitotoxic- mediated injury, further studies focused on whether sigma receptor ligands attenuate basal and NMDA-evoked NO production in vivo. Microdialysis studies of rats indicated that PPBP inhibited both basal and NMDA-evoked release of NO in the striatum. The attenuation was reversed by DuP 734, a sigma-1 receptor antagonist, adding credence to the view that the ameliorative actions of PPBP in ischemia models is via sigma-1 receptor interactions. Attenuated NO production by PPBP and like compounds may provide a possible mechanism for protection from focal ischemia. Bhardwaj, A., Sawada, M., London, E.D., Koekler, R.C., Traystman, R.J., and Kirsch, J.R. Potent Final Sigma1-receptor Ligand 4-phenyl-1- (4-phenylbutyl) Piperidine Modulates Basal and N-methyl-D-aspartate-evoked Nitric Oxide Production in Vivo. Stroke, 29(11), pp. 2404-2411, 1998; Harukuni, I., Bhardwaj, A., Traystman, R.J., Crain, B., London, E.D., and Kirsch, J.R., Neuroprotection from Focal Ischemia by 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) is Dependent on Treatment Duration in Rats. Anesth. Analg., 87(6), pp. 1299-305, 1998.

Clinical Trials Section, Treatment Branch

Broad Beneficial Effects of Two Schedules of Reinforcement for Cocaine Abstinence in Methadone Maintenance Patients

Contingency management, a treatment in which patients receive incentives for cocaine abstinence, is an effective treatment for cocaine abuse, producing significant periods of sustained abstinence in many patients. Patients receive a voucher incentive for each cocaine-free urine; vouchers have monetary values that increase with the number of consecutive cocaine-free urines. Scientists at the Intramural Research Program evaluated two different schedules of reinforcement in an effort to improve abstinence outcomes and identify the most effective procedure. Cocaine abusing methadone patients were randomly assigned to receive vouchers for 12 weeks under an escalating pay schedule, an escalating pay schedule with start-up bonuses, or a noncontingent schedule. The start-up bonuses were designed to provide substantial immediate reinforcement for initiating abstinence. Both contingent voucher interventions significantly increased subjects' longest duration of sustained cocaine abstinence, increased the percent of subjects who were cocaine abstinent and opiate abstinent across the 12 weeks of the voucher intervention, and significantly decreased self reported cocaine craving. Adding start-up bonuses did not improve abstinence outcomes and may have had an adverse effect. These results replicate the efficacy of voucher-based reinforcement of cocaine abstinence, and show that it can have broad beneficial effects as evidenced by its effects on opiate use. Silverman, K., Wong, C.J., Umbricht-Schneiter, A., Montoya, I.D., Schuster, C.R., and Preston, K.L. Broad Beneficial Effects of Two Schedules of Reinforcement for Cocaine Abstinence in Methadone Maintenance Patients. Journal of Consulting and Clinical Psychology, 66, pp. 811-824, 1998.

Cocaine Use Early in Treatment Predicts Outcome in a Behavioral Treatment Program

As part of a project to identify predictors of treatment outcome and improve patient-treatment matching, cocaine use during the first five weeks of treatment (baseline) was compared in methadone maintenance patients who had little or no periods of abstinence versus those with significant periods of sustained abstinence during the following 12 weeks of an experimental treatment. The experimental treatment was voucher-based cocaine abstinence reinforcement. Cocaine use was evaluated by qualitative and quantitative urinalysis and self-report. Abstainers (those who had successful outcomes) used significantly less cocaine in the 5-week baseline than Nonabstainers (those with less successful outcomes). Differences in cocaine use were not evident at the beginning of treatment (the first week of baseline), but Abstainers used significantly less cocaine in the fifth week of baseline compared to Nonabstainers. Qualitative urinalysis was less sensitive to baseline differences than either quantitative urinalysis or self-report. Thus, cocaine use during baseline was a critical predictor of response to the experimental treatment. Preston, K.L., Silverman, K., Higgins, S.T., Brooner, R.K., Montoya, I.D., Schuster, C.R., and Cone, E.J. Cocaine Use Early in Treatment Predicts Outcome in a Behavioral Treatment Program. J. Cons. & Clin. Psych., 66, pp. 691-696, 1998.

Pharmacotherapy Section, Treatment Branch

Cigarette Smoking During Early Cocaine Abstinence

Many cocaine addicts are heavy cigarette smokers, raising the issue of how to deal with their smoking when they enter addiction treatment and abstain from cocaine use. However, little is known about how cigarette smoking might change during early cocaine abstinence. Intramural scientists studied the number of cigarettes smoked daily (measured indirectly by computerized cigarette dispensers) by 12 cocaine-dependent research volunteers during their first 7 full days on the IRP research ward (starting about 1-1/2 days after their last cocaine use). There was no significant difference between the self-reported number of cigarettes smoked before admission and the number dispensed on the ward, nor any significant changes during the week of monitored abstinence. This finding suggests that cigarette smoking does not change significantly during early cocaine abstinence in a residential setting. Radzius, A., Gorelick, D.A. and Henningfield, J.E. Cigarette Smoking during Early Cocaine Abstinence. Am. J. Addict., 7, pp. 305-308, 1998.

Behavioral Pharmacology Section, Preclinical Pharmacology Laboratory

The GABAB Agonist Baclofen Modifies Cocaine Self-administration in Rats

In the search for a pharmacotherapy to treat cocaine addiction, most research has focused on drugs interacting directly with the dopaminergic system. Recent research, however, has indicated that alterations in other neurotransmitter systems may also be effective in altering the behavioral actions of cocaine. For example, research has shown that the GABAB agonist baclofen can alter cocaine self-administration in animals under certain conditions. IRP investigators have recently expanded on this work to show that baclofen can alter cocaine self-administration under a wider range of conditions, and that the effects of baclofen are specific to cocaine. Baclofen does not disrupt responding maintained by food reinforcement at doses that do disrupt responding maintained by cocaine. Further, drugs which affect GABAA function do not alter cocaine self-administration. Finally, authors have shown that the effects of baclofen appear to be mediated by receptors in the nucleus accumbens or ventral tegmental area. These results, along with previous work, suggest that GABAB agonists may be useful in the treatment of cocaine abuse. Shoaib, M., Swanner, L.S., Beyer, C.E., Goldberg, S.R. and Schindler, C.W. The GABAB Agonist Baclofen Modifies Cocaine Self-administration in Rats. Behavioral Pharmacology, 9, pp. 195-206, 1998.

Psychobiology Section, Preclinical Pharmacology Branch

Antagonism of Cocaine Discriminative Effects by D1-Like Dopamine Antagonists in Squirrel Monkeys

The dopamine D1 receptor antagonists have been proposed as potential treatments for cocaine abuse. IRP researchers compared the antagonism by several dopamine D1 antagonists of different behavioral effects of cocaine, each thought to be indicative of abuse liability. All of the antagonists were effective in reversing effects of cocaine, however, their effectiveness depended upon the behavior being examined. These results are consistent with previous suggestions that actions mediated by D1 receptors contribute to the effects of cocaine, and further documents the degree of involvement of D1 receptors in the various effects examined. Importantly, the predicted effectiveness of the D1 antagonists depends on which behavioral effect is examined. Therefore, assessments of treatment efficacy will require studies of the predictive validity of the various preclinical assays that are currently in use. Abstracts of the 28th Annual Meeting of the Society for Neuroscience, 24, p. 1484, 1998.

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