DAT isn't all that: cocaine reward and reinforcement require Toll-like receptor 4 signaling.

The initial reinforcing properties of drugs of abuse, such as cocaine, are largely attributed to their ability to activate the mesolimbic dopamine system. Resulting increases in extracellular dopamine in the nucleus accumbens (NAc) are traditionally thought to result from cocaine's ability to block dopamine transporters (DATs). Here we demonstrate that cocaine also interacts with the immunosurveillance receptor complex, Toll-like receptor 4 (TLR4), on microglial cells to initiate central innate immune signaling. Disruption of cocaine signaling at TLR4 suppresses cocaine-induced extracellular dopamine in the NAc, as well as cocaine conditioned place preference and cocaine self-administration. These results provide a novel understanding of the neurobiological mechanisms underlying cocaine reward/reinforcement that includes a critical role for central immune signaling, and offer a new target for medication development for cocaine abuse treatment.

Opioid activation of toll-like receptor 4 contributes to drug reinforcement.

Opioid action was thought to exert reinforcing effects solely via the initial agonism of opioid receptors. Here, we present evidence for an additional novel contributor to opioid reward: the innate immune pattern-recognition receptor, toll-like receptor 4 (TLR4), and its MyD88-dependent signaling. Blockade of TLR4/MD2 by administration of the nonopioid, unnatural isomer of naloxone, (+)-naloxone (rats), or two independent genetic knock-outs of MyD88-TLR4-dependent signaling (mice), suppressed opioid-induced conditioned place preference. (+)-Naloxone also reduced opioid (remifentanil) self-administration (rats), another commonly used behavioral measure of drug reward. Moreover, pharmacological blockade of morphine-TLR4/MD2 activity potently reduced morphine-induced elevations of extracellular dopamine in rat nucleus accumbens, a region critical for opioid reinforcement. Importantly, opioid-TLR4 actions are not a unidirectional influence on opioid pharmacodynamics, since TLR4(-/-) mice had reduced oxycodone-induced p38 and JNK phosphorylation, while displaying potentiated analgesia. Similar to our recent reports of morphine-TLR4/MD2 binding, here we provide a combination of in silico and biophysical data to support (+)-naloxone and remifentanil binding to TLR4/MD2. Collectively, these data indicate that the actions of opioids at classical opioid receptors, together with their newly identified TLR4/MD2 actions, affect the mesolimbic dopamine system that amplifies opioid-induced elevations in extracellular dopamine levels, therefore possibly explaining altered opioid reward behaviors. Thus, the discovery of TLR4/MD2 recognition of opioids as foreign xenobiotic substances adds to the existing hypothesized neuronal reinforcement mechanisms, identifies a new drug target in TLR4/MD2 for the treatment of addictions, and provides further evidence supporting a role for central proinflammatory immune signaling in drug reward.

Structure-activity relationship of trihexyphenidyl analogs with respect to the dopamine transporter in the on going search for a cocaine inhibitor.

A series of trihexyphenidyl (THP) analogs were used to search for a derivative that could serve as a cocaine inhibitor. A compound that blocks binding of the cocaine analog carboxyfluorotropane (CFT), allows dopamine uptake and exhibits low side effects could serve as a good candidate for that purpose. All analogs were tested for the extent to which they inhibit CFT binding, dopamine uptake and n-methyl scopolamine (NMS) binding. Several structure-function relationships emerged. Methylation/halogenation of THP's benzene ring enhanced the compound's ability to block CFT binding in comparison to its ability to block dopamine uptake (5a-e). Replacement of the cyclohexyl ring with a benzene ring tended to create compounds that had lower affinities to the dopamine transporter (7b compared to THP, 7d compared to 5h, 7c compared to 8c) and modification of THP's piperidine ring tended to enhance affinity to the dopamine transporter (5f-h, 8a, 8c). One analog (5f) that showed little muscarinic activity indicating that it would probably have few side effects was investigated for its effects as an in vivo cocaine inhibitor. However, it showed few antagonistic effects in vivo. Nevertheless, this work greatly elucidates the structure-function relationships required for potential cocaine inhibitors and so lays out promising directions for future research.

Novel tropane-based irreversible ligands for the dopamine transporter.

3alpha-(diphenylmethoxy)tropane (benztropine) and its analogues are tropane ring-containing dopamine uptake inhibitors that display binding and behavioral profiles that are distinct from cocaine. We previously prepared a benztropine-based photoaffinity label [125I]-(N-[4-(4'-azido-3'-iodophenyl)butyl]-3alpha-[bis(4'-fluorophenyl)methoxy]tropane, [125I]1, that covalently attached to the 1-2 transmembrane spanning region of the dopamine transporter (DAT). This was in contrast to the 4-7 transmembrane spanning region labeled by a cocaine-based photoaffinity label, [125I] 2 (RTI 82). To characterize further these different binding domains, photoaffinity ligands that had the 4'-azido-3'-iodophenyl substituent extended from the same position on the tropane ring were desirable. Thus, identification of the optimal alkyl linker between this substituent and the tropane nitrogen in the benztropine series was investigated to ultimately prepare the identical N-substituted analogue of 2. In this pursuit, the N-[4-(4'-azido-3'-iodophenyl)propyl] analogue of 3alpha-[bis(4'-fluorophenyl)methoxy]tropane (9a) was synthesized as well as two isothiocyanate analogues that do not require photoactivation (10a,b) for irreversible binding. The synthesis of these target compounds was achieved using a modification of the strategy developed for 1. Evaluation of these compounds for displacing [3H]WIN 35 428 binding at DAT in rat caudate putamen revealed that the 4'-azido-3'-iodophenylbutyl substituent, found in 1, provided optimal binding affinity and was chosen to replace the N-CH3 group on 2. Both the 4'-azido-3'-iodophenyl- and the 4'-isothiocyanatophenylbutyl analogues of 2 (25 and 26, respectively) were synthesized. Both products bound to DAT with comparable potency (IC(50) = 30 nM) to RTI 82 (2). In addition, compound 26 demonstrated wash-resistant displacement of [3H]WIN 35 428 in HEK 293 cells stably transfected with hDAT. These ligands will provide important tools for further characterizing the binding domains for tropane-based dopamine uptake inhibitors at the DAT.

[3-cis-3,5-Dimethyl-(1-piperazinyl)alkyl]-bis-(4'-fluorophenyl)amine analogues as novel probes for the dopamine transporter.

In a continuing effort to identify novel probes with which to study the dopamine transporter (DAT), we discovered that the sigma receptor antagonist, rimcazole, binds with moderate affinity (K(i)=224nM) to the DAT. The results from previous SAR studies suggested that substitution of the carbazole ring system of rimcazole with bis-(4'-fluorophenyl)amine might improve binding affinity and selectivity for the DAT. Thus, a novel series of [3-cis-3,5-dimethyl-(1-piperazinyl)alkyl]bis-(4'-fluorophenyl)amines were synthesized. The most potent compound in this series (9b) displaced [3H]WIN 35,428 binding in rat caudate-putamen (K(i)=17.6nM) with comparable affinity to GBR 12909. Despite high-affinity binding at DAT, and structural similarity to GBR 12909, preliminary studies suggest 9b behaves more like rimcazole than GBR 12909 and does not demonstrate cocaine-like psychostimulant behavior in mice.

Dopamine transporter binding without cocaine-like behavioral effects: synthesis and evaluation of benztropine analogs alone and in combination with cocaine in rodents.

RATIONALE: Previous SAR studies demonstrated that small halogen substitutions on the diphenylether system of benztropine (BZT), such as a para-Cl group, retained high affinity at the cocaine binding site on the dopamine transporter. Despite this high affinity, the compounds generally had behavioral effects different from those of cocaine. However, compounds with meta-Cl substitutions had effects more similar to those of cocaine. OBJECTIVES: A series of phenyl-ring analogs of benztropine (BZT) substituted with 3'-, 4'-, 3',4"- and 4',4"-position Cl-groups were synthesized and their pharmacology was evaluated in order to assess more fully the contributions to pharmacological activity of substituents in these positions. METHODS: Compounds were synthesized and their pharmacological activity was assessed by examining radioligand binding and behavioral techniques. RESULTS: All of the compounds displaced [3H]WIN 35,428 binding with affinities ranging from 20 to 32.5 nM. Affinities at norepinephrine ([3H]nisoxetine) and serotonin ([3H]citalopram) transporters, respectively, ranged from 259 to 5120 and 451 to 2980 nM. Each of the compounds also inhibited [3H]pirenzepine binding to muscarinic M1 receptors, with affinities ranging from 0.98 to 47.9 nM. Cocaine and the BZT analogs produced dose-related increases in locomotor activity in mice. However, maximal effects of the BZT analogs were uniformly less than those produced by cocaine, and were obtained 2-3 h after injection compared to the relatively rapid onset (within 30 min) of cocaine effects. In rats trained to discriminate i.p. saline from 29 mumol/kg cocaine (10 mg/kg), cocaine produced a dose-related increase in responding on the cocaine lever, reaching 100% at the training dose; however, none of the BZT analogs fully substituted for cocaine, with maximum cocaine responding from 20 to 69%. Despite their reduced efficacy compared to cocaine in cocaine discrimination, none of the analogs antagonized the effects of cocaine. As has been reported previously for 4'-Cl-BZT, the cocaine discriminative-stimulus effects were shifted left-ward by co-administration of the present BZT analogs. CONCLUSIONS: The present results indicate that although the BZT analogs bind with relatively high affinity and selectivity at the dopamine transporter, their behavioral profile is distinct from that of cocaine. The present results suggest that analogs of BZT may be useful as treatments for cocaine abuse in situations in which an agonist treatment is indicated. These compounds possess features such as reduced efficacy compared to cocaine and a long duration of action that may render them particularly useful leads for the development of therapeutics for cocaine abusers.

Structure-activity relationships at monoamine transporters and muscarinic receptors for N-substituted-3alpha-(3'-chloro-, 4'-chloro-, and 4',4''-dichloro-substituted-diphenyl)methoxytropanes.

The design, synthesis, and evaluation of 3alpha-(diphenylmethoxy)tropane (benztropine) analogues have provided potent and selective probes for the dopamine transporter. Structure-activity relationships (SARs) have been developed that contrast with those described for cocaine, despite significant structural similarity. Furthermore, behavioral evaluation of many of the benztropine analogues in animal models of cocaine abuse has suggested that these two classes of tropane-based dopamine uptake inhibitors have distinct pharmacological profiles. In general, the benztropine analogues do not demonstrate efficacious locomotor stimulation in mice, do not fully substitute for a cocaine discriminative stimulus, and are not appreciably self-administered in rhesus monkeys. These compounds are generally more potent than cocaine as dopamine uptake inhibitors in vitro, although their actions in vivo are not consistent with this action. These observations suggest that differing binding profiles at the serotonin and norepinephrine transporters as well as at muscarinic receptors might have significant impact on the pharmacological actions of these compounds. In addition, by varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics as well as pharmacodynamics will be directly affected. Therefore, in an attempt to systematically evaluate the impact of chemical modification on these actions, a series of N-substituted (H, CH3, allyl, benzyl, propylphenyl, and butylphenyl) analogues of 3'-chloro-, 4'-chloro-, and 4,4''-dichloro-3alpha-(diphenylmethoxy)tropanes were synthesized. These compounds were evaluated for displacement, in rat tissue, of [3H]WIN 35,428 from the dopamine transporter, [3H]citalopram from the serotonin transporter, [3H]nisoxetine from the norepinephrine transporter, and [3H]pirenzepine from muscarinic m1 receptors. SARs were developed and compared to a series of N-substituted-3alpha-(bis-4'-fluorophenyl)methoxytropanes. The present SARs followed previously reported studies with the single exception of the N-butylphenyl substituent, which did not provide the high affinity binding in any of these three sets of analogues, as it did in the 4',4''-difluoro series. X-ray crystallographic analyses of the three parent ligands (1a, 2a, and 3a) were compared to that of 3alpha-(bis-4'-fluorophenyl)methoxytropane which provided supportive evidence toward the proposal that the combination of steric bulk in both the 3-position and the N-substituent, in this class of compounds, is not optimal for binding at the dopamine transporter. These studies provide binding profile data that can now be used to correlate with future behavioral analyses of these compounds and may provide insight into the kind of binding profile that might be targeted as a potential treatment for cocaine abuse.

2D QSAR modeling and preliminary database searching for dopamine transporter inhibitors using genetic algorithm variable selection of Molconn Z descriptors.

In light of the chronic problem of abuse of the controlled substance cocaine, we have investigated novel approaches toward both understanding the activity of inhibitors of the dopamine transporter (DAT) and identifying novel inhibitors that may be of therapeutic potential. Our most recent studies toward these ends have made use of two-dimensional (2D) quantitative structure-activity relationship (QSAR) methods in order to develop predictive models that correlate structural features of DAT ligands to their biological activities. Specifically, we have adapted the method of genetic algorithms-partial least squares (GA-PLS) (Cho et al. J. Comput. -Aided Mol. Des., submitted) to the task of variable selection of the descriptors generated by the software Molconn Z. As the successor to the program Molconn X, which generated 462 descriptors, Molconn Z provides 749 chemical descriptors. By employing genetic algorithms in optimizing the inclusion of predictive descriptors, we have successfully developed a robust model of the DAT affinities of 70 structurally diverse DAT ligands. This model, with an exceptional q(2) value of 0.85, is nearly 25% more accurate in predictive value than a comparable model derived from Molconn X-derived descriptors (q(2) = 0.69). Utilizing activity-shuffling validation methods, we have demonstrated the robustness of both this DAT inhibitor model and our QSAR method. Moreover, we have extended this method to the analysis of dopamine D(1) antagonist affinity and serotonin ligand activity, illustrating the significant improvement in q(2) for a variety of data sets. Finally, we have employed our method in performing a search of the National Cancer Institute database based upon activity predictions from our DAT model. We report the preliminary results of this search, which has yielded five compounds suitable for lead development as novel DAT inhibitors.

Highly selective chiral N-substituted 3alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues for the dopamine transporter: synthesis and comparative molecular field analysis.

In a continuing effort to further characterize the role of the dopamine transporter in the pharmacological effects of cocaine, a series of chiral and achiral N-substituted analogues of 3alpha-[bis(4'-fluorophenyl)methoxy]tropane (5) has been prepared as potential selective dopamine transporter ligands. These novel compounds displaced [(3)H]WIN 35,428 binding from the dopamine transporter in rat caudate putamen with K(i) values ranging from 13. 9 to 477 nM. Previously, it was reported that 5 demonstrated a significantly higher affinity for the dopamine transporter than the parent drug, 3alpha-(diphenylmethoxy)tropane (3; benztropine). However, 5 remained nonselective over muscarinic m(1) receptors (dopamine transporter, K(i) = 11.8 nM; m(1), K(i) = 11.6 nM) which could potentially confound the interpretation of behavioral data, for this compound and other members of this series. Thus, significant effort has been directed toward developing analogues that retain high affinity at the dopamine transporter but have decreased affinity at muscarinic sites. Recently, it was discovered that by replacing the N-methyl group of 5 with the phenyl-n-butyl substituent (6) retention of high binding affinity at the dopamine transporter (K(i) = 8.51 nM) while decreasing affinity at muscarinic receptors (K(i) = 576 nM) was achieved, resulting in 68-fold selectivity. In the present series, a further improvement in the selectivity for the dopamine transporter was accomplished, with the chiral analogue (S)-N-(2-amino-3-methyl-n-butyl)-3alpha-[bis(4'-fluorophenyl)metho xy] tropane (10b) showing a 136-fold selectivity for the dopamine transporter versus muscarinic m(1) receptors (K(i) = 29.5 nM versus K(i) = 4020 nM, respectively). In addition, a comparative molecular field analysis (CoMFA) model was derived to correlate the binding affinities of all the N-substituted 3alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues that we have prepared with their 3D-structural features. The best model (q(2) = 0. 746) was used to accurately predict binding affinities of compounds in the training set and in a test set. The CoMFA coefficient contour plot for this model, which provides a visual representation of the chemical environment of the binding domain of the dopamine transporter, can now be used to design and/or predict the binding affinities of novel drugs within this class of dopamine uptake inhibitors.

Synthesis, dopamine and serotonin transporter binding affinities of novel analogues of meperidine.

A series of meperidine analogues was synthesized and the binding affinities for the dopamine and serotonin transporters were determined. The substituents on the phenyl ring greatly influenced the potency and selectivity of these compounds for the transporter binding sites. In general, meperidine (3) and its analogues were more selective for serotonin transporter binding sites and the esters 9 were more potent than the corresponding nitriles 8. The 3,4-dichloro derivative 9e was the most potent ligand of the series for dopamine transporter binding sites while the 2-naphthyl derivative 9g exhibited the most potent binding affinity and was highly selective for serotonin transporter binding sites.

Structure-activity relationships at the monoamine transporters and sigma receptors for a novel series of 9-[3-(cis-3, 5-dimethyl-1-piperazinyl)propyl]carbazole (rimcazole) analogues.

9-[3-(cis-3,5-Dimethyl-1-piperazinyl)propyl]carbazole (rimcazole) has been characterized as a sigma receptor antagonist that binds to the dopamine transporter with moderate affinity (K(i) = 224 nM). Although the binding affinities at the dopamine transporter of rimcazole and cocaine are comparable, rimcazole only depressed locomotor activity in mice and antagonized the stimulant effects produced by cocaine. The neurochemical mechanisms underlying the attenuation of cocaine's effects are not understood, although interaction at a low affinity site/state of the dopamine transporter has been suggested. To explore further this class of compounds, a series of rimcazole analogues was designed and synthesized. Displacement of [(3)H]WIN 35,428 binding at the dopamine transporter in rat caudate-putamen revealed that aromatic substitutions on rimcazole were not well tolerated, generally, with significant reductions in affinity for the 3,6-dibromo (5; K(i) = 3890 nM), 1,3, 6-tribromo (6; K(i) = 30300 nM), 3-amino (8; K(i) = 2400 nM), and 3, 6-dinitro (9; K(i) = 174000 nM) analogues. The N-phenylpropyl group was the only terminal piperazine nitrogen substituent that retained moderate affinity at the dopamine transporter (11; K(i) = 263 nM). Analogues in which the carbazole ring was replaced with a freely rotating diphenylamine moiety were also prepared. Although the diphenylamino analogue in which the terminal piperazine nitrogen was unsubstituted, as in rimcazole, demonstrated relatively low binding affinity at the dopamine transporter (24; K(i) = 813 nM), the N-phenylpropyl analogue was found to have the highest affinity for the dopamine transporter within the series (25; K(i) = 61.0 nM). All of the analogues that had affinity for the dopamine transporter inhibited [(3)H]dopamine uptake in synaptosomes, and potencies for these two effects showed a positive correlation (r(2) = 0.7731, p = 0.0018). Several of the analogues displaced [(3)H]paroxetine from serotonin transporters with moderate to high affinity, with the N-phenylpropyl derivative (11) having the highest affinity (K(i) = 44.5 nM). In contrast, none of the analogues recognized the norepinephrine transporter with an affinity of <1.3 microM. Binding affinities for sigma(1) and sigma(2) receptors were also determined, and several of the compounds were more potent than rimcazole with affinities ranging from 97 nM to >6 microM at sigma(1) sites and 145 to 1990 nM at sigma(2) sites. The compound with the highest affinity (25) at sigma(1) sites was also the compound with highest affinity at the dopamine transporter. These novel rimcazole analogues may provide important tools with which to characterize the relationship between the low affinity site or state of the dopamine transporter, sigma receptors, and their potential roles in modulating cocaine's psychostimulant actions.

Behavioral effects of cocaine: interactions with D1 dopaminergic antagonists and agonists in mice and squirrel monkeys.

The present study compared interactions among dopamine D1-like agonists and partial agonists with cocaine on the locomotor stimulant effects of cocaine, as well as the discriminative-stimulus effects of cocaine, and effects of cocaine on rates of responding. Cocaine alone produced a dose-related stimulation of locomotor activity in Swiss-Webster mice and a dose-related increase in the proportion of responses on the cocaine-appropriate response key in squirrel monkeys (Saimiri sciureus) trained to discriminate cocaine (0.3 mg/kg i.m.) from saline. None of the D1 dopaminergic agents fully reproduced these effects, with SKF 77434 producing marginal stimulation of locomotor activity and SCH 23390, SCH 39166, and SKF 77434 producing some, although incomplete substitution for cocaine in monkeys discriminating cocaine. The D1 dopamine antagonists SCH 23390, SCH 39166, and A-69024 dose-dependently shifted the cocaine dose-effect curve for locomotor activity to the right and decreased the efficacy of cocaine. The same compounds shifted the discriminative-stimulus effects of cocaine to the right without altering efficacy of cocaine. In contrast to the effects on locomotor activity, the maximal shift to the right in the discriminative-stimulus effects of cocaine was approximately 3-fold, with higher doses of the antagonists producing no greater shifts in the cocaine dose-effect curve than with intermediate doses. The partial D1 agonists (+/-)-SKF 38393, (+)-SKF 38393, and SKF 77434 also dose-dependently shifted the dose-effect curve for locomotor stimulant effects to the right and decreased the maximal effect of cocaine. These compounds only shifted the discriminative-stimulus effects of cocaine to a 2-fold maximum. In general, cocaine effects on rates of responding in the subjects discriminating cocaine from saline were only minimally antagonized by coadministration of the D1 dopaminergic agents. Both potency for producing behavioral effects alone and in antagonizing the effects of cocaine were related to binding affinities assessed by displacement of [(3)H]SCH 23390 from rat striatum. These results suggest that actions mediated by D1-like receptors contribute to the behavioral effects of cocaine. However, the various limitations to the degree of antagonism accomplished indicate that D1-like dopaminergic actions appear to be more involved in the effects of cocaine on locomotor activity, relatively less involved in the discriminative-stimulus effects of cocaine, and least involved in the effects of cocaine on operant response rates. This differential involvement of D1 dopamine receptors in these various behavioral effects of cocaine suggests problems in predicting clinical efficacy of at least D1 receptor antagonists as potential treatments for cocaine abuse. Additional studies are necessary to determine whether the antagonism of cocaine can predict therapeutic efficacy at all, and, if so, which effects when antagonized are the best predictors.

Dopamine transporter: transmembrane phenylalanine mutations can selectively influence dopamine uptake and cocaine analog recognition.

Cocaine blocks the normal role of the dopamine transporter (DAT) in terminating dopamine signaling through molecular interactions that are only partially understood. Cocaine analog structure-activity studies have suggested roles for both cationic and aromatic interactions among DAT, dopamine, and cocaine. We hypothesized that phenylalanine residues lying in putative DAT transmembrane (TM) domains were good candidates to contribute to aromatic and/or cationic interactions among DAT, dopamine, and cocaine. To test this idea, we characterized the influences of alanine substitution for each of 29 phenylalanine residues lying in or near a putative DAT TM domain. Cells express 22 mutants at near wild-type levels, manifest by DAT immunohistochemistry and binding of the radiolabeled cocaine analog [(3)H](-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (CFT). Seven mutants fail to express at normal levels. Four mutations selectively reduce cocaine analog affinities. Alanine substitutions at Phe(76), Phe(98), Phe(390), and Phe(361) located in TM domains 1 and 2, the fourth extracellular loop near TM 4 and in TM 7, displayed normal affinities for dopamine but 3- to 8-fold reductions in affinities for CFT. One TM 3 mutation, F(155)A, selectively decreased dopamine affinity to less than 3% of wild-type levels while reducing CFT affinity less than 3-fold. In a current DAT structural model, each of the residues at which alanine substitution selectively reduces cocaine analog or dopamine affinities faces a central transporter cavity, whereas mutations that influence expression levels are more likely to lie at potential helix/helix interfaces. Specific, overlapping sets of phenylalanine residues contribute selectively to DAT recognition of dopamine and cocaine.

Multiple binding sites for [125I]RTI-121 and other cocaine analogs in rat frontal cerebral cortex.

In an effort to identify novel binding sites for cocaine and its analogs, we carried out binding studies with the high-affinity and selective ligand [125I]RTI-121 in rat frontal cortical tissue. Very low densities of binding sites were found. Saturation analysis revealed that the binding was to both high- and low-affinity sites. Pharmacological competition studies were carried out with inhibitors of the dopamine, norepinephrine, and serotonin transporters. The various transporter inhibitors inhibited the binding of 15 pM [125I]RTI-121 in a biphasic fashion following a two-site binding model. The resultant data were complex and did not suggest a simple association with any single transporter. Correlational analysis supported the following hypothesis: [125I] RTI-121 binds to known transporters and not to novel sites; these include dopamine, norepinephrine, and serotonin transporters. Immunoprecipitation of transporters photoaffinity labeled with [125]RTI-82 and subsequent analysis of SDS-page gels revealed the presence of authentic dopamine transporters in these samples; displacement of the photoaffinity label occurred with a typical dopamine transporter pharmacology. These data are compatible with the binding properties of RTI-121 and the presence of several known transporters in the tissue studied.

N-substituted phenyltropanes as in vivo binding ligands for rapid imaging studies of the dopamine transporter.

Variously substituted phenyltropanes are proven as superb binding ligands for the dopamine transporter (DAT). In this study, we examine four N-substituted phenyltropanes which are derivatives of RTI-55 as in vivo binding ligands in mice. In this series, the methyl group on the nitrogen was replaced by a propyl (RTI-310), an allyl (RTI-311), a butyl (RTI-312), or a fluoropropyl (RTI-313) group. The in vitro binding potencies of these compounds at rat striatal DAT varied somewhat but were about 1 nM. While these compounds did not display marked selectivity for the dopamine transporter, they were more selective than RTI-55. Injection of the radiolabeled compound into mice resulted in striatal-to-cerebellar ratios that varied from about 4.5-6.5. The ratios peaked most rapidly for RTI-311 and RTI-313, at about 20 min. Pharmacological inhibition studies indicated that these compounds were binding to DATs in the striatum, as expected. These findings suggest that some compounds of this type may be excellent in vivo binding ligands for rapid imaging studies of the DAT.

Halogenated mazindol analogs as potential inhibitors of the cocaine binding site at the dopamine transporter.

A series of halogenated (F, Cl, Br, I), pyrimido and diazepino homologs of mazindol were prepared and evaluated for their ability to displace [3H]WIN 35,428 binding and to inhibit uptake of [3H]dopamine (DA) in rat striatal tissue. All of the compounds except for the 2'-chloro (6) and 2'-bromo (16) analogs of mazindol displaced [3H]WIN 35,428 binding and inhibited [3H]DA uptake more effectively than (R)-cocaine. Structure-activity studies indicated that best inhibition of [3H]WIN 35,428 binding occurred in the imidazo series with compounds containing one or two Cl or Br atoms in the 3'- or 4'-position of the free phenyl group. Replacement of the imidazo ring by a pyrimido or diazepino ring enhanced binding inhibition. The most potent inhibitors of [3H]WIN 35,428 binding and [3H]DA uptake were 6-(3'-chlorophenyl)-2,3,4,6-tetrahydropyrimido[2,1-alpha]isoind ol-6-ol (23; IC50 1.0 nM; 8 x mazindol) and 7-(3',4'-dichlorophenyl)-2,3,4,5-tetrahydro-7H-diazepino[2,1-alpha ]isoindol-7-ol (28; IC50 0.26 nM; 32 x mazindol), respectively. No significant differences was found between binding and uptake inhibition. Mazindol and the pyrimido and diazepino homologs 24 and 27 showed a selectivity for the DA uptake over the serotonin (5-HT) uptake site of 5-, 250-, and 465-fold, respectively, and displayed weak or no affinity for a variety of neurotransmitter receptor sites.

Highly potent cocaine analogs cause long-lasting increases in locomotor activity.

Three cocaine analogs were compared with cocaine for the capacity to affect: (1) dopamine transporter binding and function; and (2) locomotor activity. RTI-55 (3 beta-[4-iodophenyl]tropane-2 beta-carboxylic acid methyl ester tartrate), RTI-121 (3 beta-[4-iodophenyl] tropan-2 beta-carboxylic acid isopropyl ester hydrochloride) and RTI-130 (3 beta-[4-chlorophenyl-2 beta-[1,2,4-oxadiazol-3-phenyl-5-yl]tropane hydrochloride) competed for [3H]WIN 35428 binding in rat striatum in vitro, with IC50 values at least 50-fold less than that of cocaine. These analogs inhibited [3H]dopamine transport into rat striatal synaptosomes, with IC50 values again less (at least 100-fold) than that for cocaine. Intravenous RTI-55, RTI-121 or RTI-130 injection effected dose-related increases in locomotor activity in mice, with estimated relative potencies at least 10-fold greater than that of cocaine. These increases were long lasting: whereas increased activity ceased within 2 h after cocaine administration, increased locomotion was observed at least 10 h after RTI-55, RTI-121, or RTI-130 administration. Parallel line analysis indicated that the slopes of the ascending portion of the RTI-121 and RTI-130 dose-response curves differed from that of cocaine, suggesting the involvement of mechanisms different from that of cocaine.

Selective labeling of the dopamine transporter by the high affinity ligand 3 beta-(4-[125I]iodophenyl)tropane-2 beta-carboxylic acid isopropyl ester.

The iodine-125 analog of the dopaminergically selective cocaine analog 3 beta-(4-iodophenyl)tropane-2 beta-carboxylic acid isopropyl ester (RT1-121) was evaluated as a probe for the dopamine transporter in rat striatum. Saturation and kinetic studies indicated that [125I]RTI-121 binds to both high and low affinity components. The Kd of the high affinity component was 0.14 +/- 0.01 nM (mean +/- standard error), whereas the low affinity component demonstrated an affinity of 1.59 +/- 0.09 nM. The corresponding numbers of striatal binding sites labeled by [125I]RTI-121 were 295 +/- 6 and 472 +/- 59 pmol/g of tissue (original wet weight), respectively. Intrastriatal injections of 6-hydroxydopamine eliminated > 90% of specific [125I]RTI-121 binding in the striatum. The pharmacological profile of specific [125I]RTI-121 binding in the rat striatum was consistent with that of the dopamine transporter. There was a strong (r = 0.98, p < 0.0001) correlation between the potencies of drugs that displaced specific [125I]RTI-121 binding and the potencies of these drugs to inhibit the uptake of [3H]dopamine. In contrast, no correlation was found for the potencies of drugs to inhibit the uptake of either [3H]norepinephrine or [3H]serotonin. Autoradiographs produced using [125I]RTI-121 demonstrated a distribution of label consistent with the distribution of dopaminergic neurons in rat brain. Because of its high affinity and high selectivity for the dopamine transporter, [125I]RTI-121 may be an extremely useful ligand for the dopamine transporter.

Secondary amine analogues of 3 beta-(4'-substituted phenyl)tropane-2 beta-carboxylic acid esters and N-norcocaine exhibit enhanced affinity for serotonin and norepinephrine transporters.

N-Norcocaine (2) and six N-nor-3 beta-(4'-substituted phenyl)tropane-2 beta-carboxylic acid esters (4a-f) were synthesized by N-demethylation of cocaine (1) and the appropriate 3 beta-(substituted phenyl)-tropane analogues (3a-f) with alpha-chloroethyl chloroformate. Radioligand binding affinities of 2 and 4a-f at the DA, 5-HT, and NE transporter were measured and compared to those of 1 and 3a-f. N-Demethylation produced relatively small effects at the DA transporter. In contrast, 4-19-fold and 2-44-fold enhanced affinity at the serotonin and norepinephrine transporter resulted from demethylation. N-Nor-3 beta-(4'-iodophenyl)tropane-2 beta-carboxylic acid methyl ester (4d) with an IC50 = 0.36 nM showed the greatest affinity for the serotonin transporter. However, N-nor-3 beta-(4'-ethylphenyl)tropane-2 beta-carboxylic acid methyl ester (4e) showed the greatest selectivity for the serotonin transporter.

Ion channel inhibitors may function as potential modulators of cocaine binding.

The effects of NaCl, KCl and Ca2Cl were determined in a binding assay, using the analog of cocaine [3H]WIN 35,428. The addition of NaCl had no effect upon specific binding; however, Ca2Cl and to a lesser extent, KCl decreased binding. Various Na+, K+, Cl- and Ca2+ channel antagonists were tested for their ability to inhibit specific binding of [3H]WIN 35,428. Most of the Na+ channel blockers tested were of moderate affinity, the exceptions being benzamil and flunarizine. Both of these agents also show activity at the Ca2+ channel. The K+ channel blockers were of low and moderate affinity, while the Cl- channel blockers had no effect. Of the Ca2+ channel blockers tested, only pimozide demonstrated a high affinity. This was postulated to be due to its ability to act upon both L and T-type channels. These results suggest that the Ca2+ channel blockers deserve further study as potential useful therapeutic agents in the treatment of cocaine addiction.