Dorota Matecka

Doses of GBR12909 that suppress cocaine self‐administration in non‐human primates substantially occupy dopamine transporters as measured by [11C] WIN35,428 PET scans

GBR12909 (GBR) is a high‐affinity, selective, and long‐acting inhibitor of dopamine (DA) uptake that produces a persistent and noncompetitive blockade of DA transporters and substantially reduces cocaine‐induced increases in extracellular DA in the nucleus accumbens of rats. Prior studies showed that intravenous infusion of GBR to Rhesus monkeys selectively reduced (1 mg/kg) and eliminated (3 mg/kg) cocaine self‐administration. This study tested the hypothesis that doses of GBR that reduce cocaine self‐administration in nonhuman primates produce significant occupation of DA transporters. DA transporters were quantitated in two baboons using [11C]WIN35,428 and positron emission tomography (PET). Each baboon underwent paired control/blocked PET scans (performed on three separate study days, 3–4 weeks apart). On the first scan the baboon received saline (3 ml/kg) 90 minutes before the injection of the radiotracer. GBR (1 mg/kg i.v.) was infused 90 minutes before the second [11C]WIN 35,428 study. The same experimental design was repeated with GBR doses of 3 and 10 mg/kg, respectively. Doses of 1 (n = 2), 3 mg/kg (n = 2), and 10 mg/kg (n = 2) reduced binding potential by 26, 53, and 72%, respectively. GBR was well tolerated in all baboons. These results demonstrate that doses of GBR that suppress cocaine self‐administration in nonhuman primates also produce high occupancy of the DA transporter. These data strongly suggest that occupancy for the DA transporter by GBR explains its ability to attenuate cocaine‐induced increases in extracellular DA and to suppress cocaine self‐administration. Moreover, these data suggest that experimental human studies of orally administered GBR to test the DA hypothesis of cocaine addiction should use doses that produce at least 70% occupancy of the DA transporter. Synapse 32:44–50, 1999.

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. This hypothesis has not been adequately tested in humans. Previous in vivo rodent microdialysis demonstrated that the high affinity DA uptake inhibitor, GBR12909, attenuates cocaine‐ and amphetamine‐induced increases in mesolimbic DA. The present study determined the ability of GBR12909 to attenuate amphetamine‐induced increases in striatal DA as measured by [11C]raclopride continuous infusion positron emission tomography (PET) scans in two Papio anubis baboons. [11C]Raclopride was given in a continuous infusion paradigm resulting in a flat volume of distribution vs. time for up to 45 min postinjection. At that time, 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 22–41%. GBR12909 (1 mg/kg, slow i.v. infusion) was administered 90 min before the administration of the radiotracer. The comparison of the volume of distribution before and after administration of GBR12909 showed that GBR12909 inhibited amphetamine‐induced DA release by 74%. 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. Furthermore, this quantitative approach demonstrates a way of testing various treatment medications, including other forms of GBR12909 such as a decanoate derivative. Synapse 33:268–273, 1999.

Asp147 in the third transmembrane helix of the rat μ opioid receptor forms ion-pairing with morphine and naltrexone

We tested the hypotheses that the carboxylate side chain of Asp147 of the mu opioid receptor interacts with the protonated nitrogen of naltrexone and morphine and that this interaction is important for pharmacological properties of the two compounds. Mutation of Asp147 to Ala or Asn substantially reduced the affinity of naltrexone and the affinity, potency and efficacy of morphine, while the Glu mutant had similar properties as the wildtype, indicating the significant role of the carboxylate group of Asp147 in receptor binding and activation. This role could be due to its direct interaction with ligands or involvement in interhelical interactions. The unprotonated analogs of naltrexone and morphine, cyclopropylcarbonyl noroxymorphone (CPCNOM) and N-formylnormorphine (NFNM), respectively, were used to discriminate between these mechanisms. CPCNOM was much less potent as an antagonist and had substantially lower affinity for the mu receptor than naltrexone. Similarly, NFNM was unable to activate the mu receptor and had much lower affinity than morphine. These results indicate the importance of the protonated nitrogen. Notably, the D147A and D147N mutations did not appreciably affect the binding affinities of CPCNOM and NFNM. In addition, the D147E mutant had similar affinities for CPCNOM and NFNM as the D147A and D147N mu receptors. Thus, the carboxylate group of Asp147 is not important for binding of the two unprotonated compounds. These results indicate that the carboxylate group of Asp147 of the mu receptor interacts directly with the protonated nitrogen of naltrexone and morphine and this interaction is important for binding and receptor activation.

Phentermine/fenfluramine decreases cocaine self-administration in rhesus monkeys.

DOPAMINERGIC agonists can decrease cocaine selfadministration at doses that do not decrease food-maintained responding, a pre-clinical effect indicative of a potential treatment for human cocaine abuse. To assess whether similar effects could be obtained with medications currently used to treat substance abuse, phentermine and fenfluramine were given alone and in combination to rhesus monkeys responding under schedules of food and cocaine delivery. Phentermine decreased cocaine-maintained responding with no effect on food-maintained responding. Fenfluramine also selectively decreased cocaine-maintained responding, but only at the highest dose. Combining a lower dose of fenfluramine with phentermine selectively decreased cocaine-maintained responding, but not more than with phentermine alone. These results suggest that phentermine, as well as its combination with fenfluramine, may be useful in the treatment of cocaine abuse.

Cocaine cross-sensitization to dopamine uptake inhibitors: Unique effects of GBR12909

Repeated administration of cocaine will cross-sensitize the locomotor response to a variety of psychomotor stimulants. The ability of cocaine to cross-sensitize the locomotor effects of other psychomotor stimulants provides information relevant to the pharmacological mechanisms underlying the sensitization process. The purpose of the current experiment was to investigate the ability of cocaine to cross-sensitize the locomotor effects of several dopamine uptake blockers with unique pharmacological profiles. Cocaine (40 mg/kg, IP) or saline was administered prior to a locomotor session on day one. On day 2, a full dose-effect curve was established for the locomotor effects of cocaine, RTI-55, mazindol, and GBR12909. Previous exposure to cocaine significantly affected locomotor activity and stereotopy-like behavior produced by cocaine, mazindol, RTI-55, and GBR12909. However, GBR12909 was unique in that the maximal stimulant effect and slope of the dose-effect curve was significantly depressed and the stereotopy-like behavior was unchanged. Thus, despite the similarity of these compounds in their ability to inhibit dopamine uptake, cocaine-induced sensitization did not generalize to GBR12909. This study further demonstrates the unique pharmacology of GBR12909 and supports the further study of this compound as a potential treatment medication for cocaine abuse.

Pharmacologic actions of subtype-selective and novel GABAergic ligands in rat lines with differential sensitivity to ethanol

Alcohol-nontolerant (ANT) rats, produced by selective breeding for high sensitivity to motor-impairing effects of ethanol, have a point mutation in the cerebellar gamma-aminobutyric acid type A (GABAA) receptor alpha 6 subunit, which has been proposed to underlie enhanced sensitivity to benzodiazepine agonists as well. We compared ANT and alcohol-tolerant (AT) rats using behavioral and neurochemical methods to assess the significance of alpha 6- and non alpha 6-containing GABAA receptor subtypes. Motor performance in a tilting plane test was largely unaffected by a type I benzodiazepine receptor-preferring agonist, zolpidem [1-10 mg/kg, intraperitoneally (IP)], partial benzodiazepine agonists bretazenil and ZG-63 (both at 40 mg/kg, IP), and a novel broad-spectrum anticonvulsant loreclezole (40 mg/kg, IP) in both ANT and AT rats. In contrast, diazepam (10 mg/kg, IP) impaired performance of the ANT but not AT animals. These data, supported by results from brain regional autoradiography of [3H]Ro15-4513 and membrane binding of [3H]ZG-63 and [35S]TBPS as influenced by these ligands, strongly suggest that only ligands with full agonist actions on mutant (ANT) but not wild-type (AT) alpha 6-containing GABAA receptors are able to produce motor impairment in the ANT rats.

Studies of the biogenic amine transporters. VII. Characterization of a novel cocaine binding site identified with [125I]RTI‐55 in membranes prepared from human, monkey and guinea pig caudate

[125I]RTI‐55 is a cocaine analog with high affinity for dopamine (DA) and serotonin (5‐HT) transporters. Quantitative ligand binding studies revealed a novel high affinity [125I]RTI‐55 binding site assayed under 5‐HT transporter (SERT) conditions which has low affinity for almost all classic biogenic amine transporter ligands, including high affinity 5‐HT transporter inhibitors such as paroxetine, but which retains high affinity for cocaine analogs. This site, termed SERTsite2 for its detection under 5‐HT transporter conditions (not for an association with the SERT) occurs in monkey caudate, human caudate, and guinea pig caudate membranes, but not in rat caudate membranes. SERTsite2 is distinguished from the DA transporter (DAT) and SERT by several criteria, including a distinct ligand‐selectivity profile, the inability to detect SERTsite2 in cells stably expressing the cloned human DAT, and insensitivity to irreversible ligands which inhibit [125I]RTI‐55 binding to the DAT and SERT. Perhaps the most striking finding about SERTsite2 is that a wide range of representative antidepressant agents have very low affinity for SERTsite2. The affinity of cocaine for this site is not very different from the concentration cocaine achieves in the brain at pharmacological doses. Viewed collectively with the observation that ligands with high affinity for SERTsite2 are mostly cocaine analogs, these data lead us to speculate that actions of cocaine which differ from those of classic biogenic amine uptake inhibitors may be mediated in part via SERTsite2. Synapse 28:322–338, 1998. Published 1998 Wiley‐Liss, Inc.

Opioid peptide receptor studies. 4. Antisense oligodeoxynucleotide to the delta opioid receptor delineates opioid receptor subtypes

Prior work in our laboratory has identified putative subtypes of delta (delta cx-1, delta cx-2, delta ncx-1, delta ncx-2) and kappa 2 (kappa 2a and kappa 2b) receptors. Previous studies showed that chronic (three day) i.c.v. administration of antisense oligodeoxynucleotide to the cloned delta opioid receptor selectively decreased [3H][D-Ala2,D-Leu5]enkephalin binding to the delta ncx site, not the delta cx-2 site. The present study extends this work by demonstrating that delta antisense DNA selectively affects the delta ncx-2 site sparing the other putative delta receptor subtypes and kappa 2 receptor subtypes. This selectivity is not due to anatomically specific effects of delta antisense DNA since autoradiograms show that delta binding is reduced in all regions of the brain after chronic i.c.v. administration of delta antisense DNA. These data strongly suggest that the delta cx-1, delta cx-2, delta ncx-1, kappa 2a and kappa 2b binding sites are different proteins than the delta ncx-2 binding site, which, based on its sensitivity to delta antisense DNA, is synonymous to the cloned delta opioid receptor. Viewed collectively, these data suggest that administration of delta antisense DNA, and by extension other receptor-selective antisense DNA, is a powerful approach to distinguishing between postulated receptor subtypes.

Opioid Peptide Receptor Studies. 8. One of the Mouse Brain δNCX Binding Sites is Similar to the Cloned Mouse Opioid δ Receptor: Further Evidence for Heterogeneity of δ Opioid Receptors

Abstract Xu, H., Y-F. Lu, J. S. Partilla, J. Pinto, S. N. Calderon, D. Matecka, K. C. Rice, J. Lai, F. Porreca, S. Ananthan and R. B. Rothman. Opioid peptide receptor studies. 8. One of the mouse brain δNCX binding sites is similar to the cloned moused opioid δ receptor: further evidence for heterogeneity of δ opioid receptors. Peptides 19(2) 343–350, 1998.—Quantitative ligand binding studies resolved two subtypes of the δ opioid receptor, termed δncx1 and δncx2, in mouse brain membranes depleted of μ receptors by pretreatment with the irreversible ligand, BIT. The purpose of the present study was to compare the binding parameters, ligand-selectivity profile and pharmacological properties of the cloned mouse δ receptor (MDOR) stably expressed in a cell line to the δncx binding sites of mouse brain. [3H][D-Ala2,D-Leu5]enkephalin labeled a single binding site in membranes prepared from MDOR cells under several different assay conditions including BIT-pretreatment. The MDOR had high affinity for δ agonists and antagonists. [3H][D-Ala2,D-Leu5]enkephalin labeled two binding sites in mouse brain membranes depleted of μ receptors by pretreatment with BIT: the δncx1 site (high affinity for DPDPE and deltorphin) and the δncx2 site (low affinity for DPDPE and deltorphin). Some agents were moderately selective for the deltancx2 site: [pCl]DPDPE (10.9-fold), JP41 (5.9-fold) and JP45 (3.8-fold). The Ki values of 12 opioids at the mouse MDOR were determined. These values were highly correlated with their values at the deltancx1 site but not the deltancx2 site. These data suggest that the deltancx2 site may be distinct from the cloned delta opioid receptor.

Opioid peptide receptor studies. 9. Identification of a novel non-μ- non-δ-like opioid peptide binding site in rat brain

Abstract The two binding sites had lower (δ ncx-2 , Ki = 96.6 nM) and higher (δ ncx-1 , Ki = 1.55 nM) affinity for DPDPE. The ligand-selectivity profile of the δ ncx-1 site was that of a classic δ binding site. The ligand-selectivity profile of the δ ncx-2 site was neither μ- or δ-like. The Ki values of selected agents for the δ ncx-2 site were: [pCl]DPDPE (3.9 nM), DPLPE (140 nM), and DAMGO (2.6 nM). Under these assay conditions, [ 3 H][ d -Ala 2 , d -Leu 5 ]enkephalin binding to the cells expressing the cloned μ receptor is very low and pretreatment of cell membranes with BIT almost completely inhibits [ 3 H]DAMGO and [ 3 H][ d -Ala 2 , d -Leu 5 ]enkephalin binding. Intracerebroventricular administration of antisense DNA to the cloned delta receptor selectively decreased [ 3 H][ d -Ala 2 , d -Leu 5 ]enkephalin binding to the δ ncx-1 site. Administration of buprenorphine to rats 24 h prior to preparation of membranes differentially affected μ, δ ncx-1 , and δ ncx-2 binding sites. Viewed collectively, these studies have identified a novel non-μ- non-δ-like binding site in rat brain.