The focus of this research is to better understand pharmacological mechanisms underlying the behavioral effects of cocaine that lead to its abuse. Further, the mechanistic information obtained can be used to direct further research towards the discovery of novel chemical entities that may serve as medical treatments for cocaine abuse. There is a large unmet medical need for medical treatments for cocaine addiction. The primary biological target of cocaine is thought to be the dopamine transporter (DAT), which functions to terminate dopamine (DA) neurotransmission through translocation of the neurotransmitter from the extracellular to the intracellular space. By interfering with this process cocaine indirectly increases the actions of DA at its various receptors. This DAT hypothesis of the actions of cocaine suggests that any agent that interferes with DA uptake into the cell will have effects like those of cocaine, and most if not all of the traditionally studied drugs acting at the DAT share pharmacological effects with those of cocaine. Analogs of benztropine (BZT) also bind with high affinity to the DAT. However, the BZT analogs are distinct from cocaine in their behavioral activity. Because of these pharmacological differences, and the potential of BZT analogs to serve as medical treatments for cocaine abuse, we have extensively studied compounds from this chemical group. Among BZT analogs previously studied are several N-substituted analogs with high affinity for the DAT and varying degrees of selectivity for the DAT over other targets. The N-butyl analog of BZT (JHW 007) was studied in detail because it was substantially less efficacious than cocaine in producing various cocaine-like effects in laboratory animals. Further, JHW 007 was the first BZT analog that was demonstrated to antagonize the psychomotor stimulant effects of cocaine in laboratory animals. In comparison, standard DAT inhibitors, such as methylphenidate, typically potentiate the effects of cocaine. Previous studies of BZT analogs, have investigated several off-target actions (those not mediated by the DAT) as potentially responsible for their differences from cocaine. Studies of the effects of BZT analogs at M1 muscarinic and H1 histaminic receptors suggested that these off-target sites of action could not fully account for the differences between BZT analogs and cocaine. Studies reported during the past year suggest that differences in pharmacology of BZT analogs and standard DAT inhibitors likely stem from fundamental differences between the manner in which they interact with the DAT. Several studies of combinations of BZT analogs with cocaine were undertaken to characterize the functional consequences of the differences in binding conformation equilibrium induced by cocaine and BZT analogs. To determine sites responsible for the cocaine-antagonist effects of JHW 007, its in vitro binding was compared to that of the standard cocaine-like DAT inhibitor, WIN 35,428. A one-site binding model best fit the data for binding of 3HWIN 35,428 data. In contrast, the data for the binding of 3HJHW 007 binding was best fit by a two-site model. However, a one-site fit was observed with membranes from a cell line transfected with the human DAT. Drugs selective for the norepinephrine and serotonin transporters had relatively low affinity in competition with 3HJHW 007 binding, as did drugs selective for other sites previously identified as potential JHW 007 binding sites. The association of 3HWIN 35,428 best fit a one-phase model, whereas the association of 3HJHW 007 best fit a two-phase model in all tissues. Because cocaine-antagonist effects of JHW 007 have been previously observed soon after injection, its rapid association observed here may contribute to those effects. Multiple 3HJHW 007 binding sites were obtained in tissue from mice lacking the DAT, suggesting these as yet unidentified sites as potential contributors to the cocaine-antagonist effects of JHW 007. Unlike WIN 35,428, the binding of JHW 007 was Na+-independent. Thus JHW 007 binds to multiple sites some of which are not related to the DAT. In addition, the sodium independence of JHW 007 binding suggests that its interaction with the DAT differs from that of standard cocaine-like DAT inhibitors. The DAT independent binding and the novel interaction of JHW 007 with the DAT may contribute to its antagonism of the behavioral effects of cocaine. Previous studies suggested that differences between the behavioral effects of cocaine and analogs of benztropine were related to the relatively slow onset of action of the latter compounds. Several N-substituted analogs of benztropine (GA 1-69, GA 2-50, GA 2-99, JHW 013) with relatively fast onsets of effect were studied to assess whether that kinetic profile would render the effects more similar to those of cocaine. Only one of the compounds (JHW 013) increased locomotor activity, and the increases were modest compared with those of cocaine. In rats trained to discriminate cocaine from saline none of the compounds produced full substitution for cocaine. None of the compounds produced reinforcing effects assessed with place-conditioning, indicating minimal abuse liability. The compounds had nM affinities at the DAT, with uniformly lower affinities at norepinephrine and serotonin transporters. Affinities at muscarinic M1 receptors were from 100- to 300-fold lower than DAT affinities, suggesting minimal contribution of those sites to the behavioral effects of the compounds. Affinities at histaminic H1 sites were from 11- to 43-fold lower than those for the DAT. The compounds also had affinity for sigma, 5-HT1, and 5-HT2 receptors that may have contributed to their behavioral effects. Together, the results indicate that a slow onset of action is not a necessary condition for reduced cocaine-like effects of atypical DAT ligands and suggest several mechanisms that may contribute to the reduced cocaine-like efficacy of these compounds. In addition to their potential use as treatments for cocaine abuse, atypical DAT inhibitors such as the BZT analogs may have use in the treatment of attention deficit hyperactivity disorder (ADHD). Specifically, medicines lacking abuse liability, such as the BZT analogs, may provide improvements in safety over some of the medications currently in use. Preclinical models for assessing efficacy in the treatment of ADHD, such as the 5-Choice Serial Reaction-Time (5-CSRT) task, have been problematic in that most show small effects when the results are positive, as well as variable responses across subjects which renders positive effects difficult to discern. We modified the task by training subjects to respond with pay-off ratios of 1, 3, or 10 for correct detections of an infrequently presented stimulus. Significant increases in accuracy of signal detection under the modified 5-CSRT task were obtained when each 3 or 10 responses were reinforced. In addition, clinical observations suggest that patients have increased failure of attention when reinforcement is intermittent adding to the content validity of this test. Thus, the results with these N-substituted BZT analogs suggest the possibility for their development as medical treatments for cocaine abuse and the likelihood that the compounds themselves will have low liability for abuse. More generally, the present results confirm that the DAT can serve as an important target for the discovery of compounds with potential for development as cocaine abuse medical treatments.
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