The biological target of cocaine is thought to be the dopamine transporter (DAT), which terminates dopamine (DA) neurotransmission. The DAT hypothesis of the actions of cocaine suggests that any agent that interferes with DA uptake will have effects like those of cocaine, and most if not all of the traditionally studied drugs acting at the DAT have effects consistent with the hypothesis. Analogs of benztropine (BZT) also bind with high affinity to the DAT. However, BZT analogs are distinct from cocaine in many effects including stimulation of DA neurotransmission, locomotor stimulation, subjective and reinforcing effects. Among BZT analogs studied are several N-substituted analogs, including the N-butyl analog (JHW007) which antagonized the locomotor-stimulant effects of cocaine. Standard DAT inhibitors potentiate the effects of cocaine. Mechanistic hypotheses regarding: 1) the reduced cocaine-like efficacy of BZT analogs, or 2) the cocaine-antagonist effects involve pharmacokinetics, off-target (non-DAT) sites, and actions at the DAT. PK hypothesis. All of the BZT analogs studied were found at high concentrations in brain within minutes after injection. Levels in brain of BZT analogs ranged from 4-15 g/g of tissue (≈3-3 μM, depending on the compound) and exceeded Ki values (11-30 nM) for DAT binding. Thus, PK studies did not support poor CNS penetration as a cause of the lower cocaine-like efficacy of BZT analogs. Off-Target Sites. Binding studies have shown that most BZT analogs are selective for DAT, compared to other monoamine transporters. However like BZT itself, many have affinity for M1 and H1 receptors. Studies on the binding and in vivo pharmacology of BZT analogs indicate that H1 or M1 receptor actions contribute little, if anything, to the lower efficacy or cocaine-antagonist effects of BZT analogs. Studies of the radiolabeled cocaine antagonist, JHW007, showed that it has high affinity for the DAT, and binds to a single site in DAT transfected cell membranes, but multiple sites in rodent native tissue, including that from DAT knockout mice. The non-DAT sites were not sensitive to norepinephrine or serotonin transport inhibitors, nor to drugs selective for several sites previously identified as potential off-target sites of JHW007. Several other targets have been identified as potentially contributing to the atypical DAT inhibitory effect of BZT analogs. Among those targets are sigma (σ), cannabinoid CB1, 5-HT1, and 5-HT2 receptors. The potential contribution of these sites to the reduced cocaine-like or cocaine-antagonist effects of BZT analogs is currently under study. DAT hypotheses. Structure-activity studies suggested that BZT analogs bind to the DAT differently from cocaine and it congeners, which were supported by site-directed mutagenesis studies. Photoaffinity labeling studies also support the hypothesis of different binding domains for cocaine analogs compared to BZT analogs. Additionally, studies of the reaction of methanethiosulfonate ethyltrimethyl-ammonium (MTSET) to various cysteine residues within the human DAT suggest differences between BZT and cocaine in their binding to different DAT conformations. In a previous study hDAT was point mutated by replacing the two external endogenous cysteines to alanines (C90A-C306A). Additionally, the I159 was mutated to cysteine which would be accessible to MTSET when the DAT assumes an outward- but not inward-facing conformation. The sulfhydral reactive and positively charged MTSET reacts with cysteine residues and inactivates the transporter. Cocaine potentiated the DAT inhibition produced by MTSET alone, consistent with the induction of a DAT conformation open to the extracellular environment, and thus MTSET. In contrast, BZT analogs protected against the DAT inhibition produced by MTSET, suggesting that these compounds stabilize the DAT in a closed conformation. Studies with a mutation of the Tyr335 which is hypothesized as critical for regulating the conformational equilibrium of the DAT, showed impaired DA transport and 100-fold decreased potency of cocaine. In contrast, BZT analogs were only 7- to 58-fold less potent against the Y335A mutant, consistent with the difference in conformational preference between BZT and cocaine analogs. Further, a good relationship between the potency shift due to the DAT mutation and the behavioral effectiveness of BZT analogs was found. Finally, the DAT binding of JHW007, unlike that of cocaine-like drugs, was Na+-independent. This feature of JHW007 has been linked to the conformational status of the DAT, which contributes to the antagonism of cocaine. All of these studies together suggest that the conformational preference of DAT ligands can induce a dynamic change in functionality of the DAT, which is consistent with neurochemical and behavioral outcomes. Recently completed studies showed that the antagonism of cocaine stimulant effects was directly related to JHW007 dose, and that JHW007 was an effective antagonist of the DA uptake inhibitor GBR 12909, but not of SNC-80, a delta-opioid agonist that stimulates activity through non-dopaminergic mechanisms. Though maximal in vivo DAT occupancy by JHW007 was obtained over 4 hours after injection, antagonist effects were obtained within 10 min after injection. The antagonism shortly after injection may be related to the initial fast phase association obtained in the in vitro binding studies with JHW007. As yet unpublished studies indicate that ten N-substituted BZT analogs in addition to JHW007 have cocaine-antagonist effects in vivo. Several of these compounds have rapid onsets of behavioral effects, consistent with the finding with JHW007 that its slow onset of action is not necessary for its cocaine antagonist effects. Another study compared the effects of pretreatment with N-substituted BZT analogs and other monoamine uptake inhibitors on the self-administration of cocaine. In that study, pre-session treatments with methylphenidate shifted the dose-effect curve for cocaine self-administration leftward, indicating a potentiation of the effects of cocaine. In contrast, the BZT analogs, AHN 2-005 and JHW007 each shifted the cocaine dose-effect curve downward, indicating an antagonism of the reinforcing effect of cocaine. Both AHN 2-005 and JHW007 blocked cocaine self administration at doses that had no effects on food-maintained responding. The cocaine-antagonist effects of JHW007 suggest the interaction between JHW007 and cocaine might involve alteration of cocaine-induced stimulation of extracellular DA levels. Therefore, the effects of combinations of different doses of JHW007 and cocaine on stimulation of DA levels in the NAc shell were compared with combinations of cocaine and the standard DAT blocker, WIN35428. Increases in extracellular DA produced by cocaine-WIN35428 combinations were greater than that produced by cocaine alone, and also greater than the predicted dose-additive effect. In contrast, the effects of cocaine-JHW007 combinations were consistently less than additive, and shortly after injection JHW007 antagonized the effects of cocaine on NAc shell DA levels. These results suggest that JHW007 may reduce the effectiveness of cocaine in stimulating dopamine neurotransmission in brain areas related to its reinforcing actions. Other Therapeutic Applications. Drugs that act at the DAT also have therapeutic utility in the treatment of ADHD. We have used the 5-choice serial reaction time task (5-CSRTT) as a validated model system to assess the potential of dopamine uptake inhibitors as treatments for ADHD. Preliminary studies indicated that the BZT analog AHN 2-005 increases attention under this task.
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