Synthesis and Pharmacological Evaluation of Enantiopure N-Substituted Ortho-c Oxide-Bridged 5-Phenylmorphans.

The design of enantiopure stereoisomers of N-2-phenylcyclopropylmethyl-substituted ortho-c oxide-bridged phenylmorphans, the E and Z isomers of an N-cinnamyl moiety, and N-propyl enantiomers were based on combining the most potent oxide-bridged phenylmorphan (the ortho-c isomer) with the most potent N-substituent that we previously found with a 5-(3-hydroxy)phenylmorphan (i.e., N-2-phenylcyclopropyl methyl moieties, N-cinnamyl, and N-propyl substituents). The synthesis of the eight enantiopure N-2-phenylcyclopropylmethyl ortho-c oxide-bridged phenylmorphans and six additional enantiomers of the N-substituted ortho-c oxide-bridged phenylmorphans (N-E and Z-cinnamyl compounds, and N-propyl compounds) was accomplished. The synthesis started from common intermediates (3R,6aS,11aS)-10-methoxy-1,3,4,5,6,11a-hexahydro-2H-3,6a-methano-benzofuro[2,3-c]azocine (+)-6 and its enantiomer, (3S, 6aR, 11aR)-(-)-6, respectively. The enantiomers of ±-6 were obtained through salt formation with (S)-(+)- and (R)-(-)-p-methylmandelic acid, and the absolute configuration of the (R)-(-)-p-methylmandelate salt of (3S, 6aR, 11aR)-(-)-6 was determined by single-crystal X-ray analysis. The enantiomeric secondary amines were reacted with N-(2-phenylcyclopropyl)methyl derivatives, 2-(E)-cinnamyl bromide, and (Z)-3-phenylacrylic acid. These products led to all of the desired N-derivatives of the ortho-c oxide-bridged phenylmorphans. Their opioid receptor binding affinity was measured. The compounds with MOR affinity < 50 nM were examined for their functional activity in the forskolin-induced cAMP accumulation assay. Only the enantiomer of the N-phenethyl ortho-c oxide-bridged phenylmorphan ((-)-1), and only the (3S,6aR,11aR)-2-(((1S,2S)-2-phenylcyclopropyl)methyl)-1,3,4,5,6,11a-hexahydro-2H-3,6a-methanobenzofuro[2,3-c]azocin-10-ol isomer ((+)-17), and the N-phenylpropyl derivative ((-)-25) had opioid binding affinity < 50 nM. Both (-)-1 and (-)-25 were partial agonists in the cAMP assay, with the former showing high potency and low efficacy, and the latter with lower potency and less efficacy. Most interesting was the N-2-phenylcyclopropylmethyl (3S,6aR,11aR)-2-(1S,2S)-enantiomer ((+)-17). That compound had good MOR binding affinity (Ki = 11.9 nM) and was found to have naltrexone-like potency as a MOR antagonist (IC50 = 6.92 nM).

Dopamine Transporter Dynamics of N-Substituted Benztropine Analogs with Atypical Behavioral Effects.

Atypical dopamine transporter (DAT) inhibitors, despite high DAT affinity, do not produce the psychomotor stimulant and abuse profile of standard DAT inhibitors such as cocaine. Proposed contributing features for those differences include off-target actions, slow onsets of action, and ligand bias regarding DAT conformation. Several 3α-(4',4''-difluoro-diphenylmethoxy)tropanes were examined, including those with the following substitutions: N-(indole-3''-ethyl)- (GA1-69), N-(R)-2''-amino-3''-methyl-n-butyl- (GA2-50), N-2''aminoethyl- (GA2-99), and N-(cyclopropylmethyl)- (JHW013). These compounds were previously reported to have rapid onset of behavioral effects and were presently evaluated pharmacologically alone or in combination with cocaine. DAT conformational mode was assessed by substituted-cysteine accessibility and molecular dynamics (MD) simulations. As determined by substituted-cysteine alkylation, all BZT analogs except GA2-99 showed bias for a cytoplasmic-facing DAT conformation, whereas cocaine stabilized the extracellular-facing conformation. MD simulations suggested that several analog-DAT complexes formed stable R85-D476 "outer gate" bonds that close the DAT to extracellular space. GA2-99 diverged from this pattern, yet had effects similar to those of other atypical DAT inhibitors. Apparent DAT association rates of the BZT analogs in vivo were slower than that for cocaine. None of the compounds was self-administered or stimulated locomotion, and each blocked those effects of cocaine. The present findings provide more detail on ligand-induced DAT conformations and indicate that aspects of DAT conformation other than "open" versus "closed" may facilitate predictions of the actions of DAT inhibitors and may promote rational design of potential treatments for psychomotor-stimulant abuse.

The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats.

The dopamine transporter (DAT) regulates dopamine (DA) neurotransmission by recapturing DA into the presynaptic terminals and is a principal target of the psychostimulant cocaine. The sigma-1 receptor (σ1R) is a molecular chaperone, and its ligands have been shown to modulate DA neuronal signaling, although their effects on DAT activity are unclear. Here, we report that the prototypical σ1R agonist (+)-pentazocine potentiated the dose response of cocaine self-administration in rats, consistent with the effects of the σR agonists PRE-084 and DTG (1,3-di-o-tolylguanidine) reported previously. These behavioral effects appeared to be correlated with functional changes of DAT. Preincubation with (+)-pentazocine or PRE-084 increased the Bmax values of [3H]WIN35428 binding to DAT in rat striatal synaptosomes and transfected cells. A specific interaction between σ1R and DAT was detected by co-immunoprecipitation and bioluminescence resonance energy transfer assays. Mutational analyses indicated that the transmembrane domain of σ1R likely mediated this interaction. Furthermore, cysteine accessibility assays showed that σ1R agonist preincubation potentiated cocaine-induced changes in DAT conformation, which were blocked by the specific σ1R antagonist CM304. Moreover, σ1R ligands had distinct effects on σ1R multimerization. CM304 increased the proportion of multimeric σ1Rs, whereas (+)-pentazocine increased monomeric σ1Rs. Together these results support the hypothesis that σ1R agonists promote dissociation of σ1R multimers into monomers, which then interact with DAT to stabilize an outward-facing DAT conformation and enhance cocaine binding. We propose that this novel molecular mechanism underlies the behavioral potentiation of cocaine self-administration by σ1R agonists in animal models.

2-Substituted 3ß-Aryltropane Cocaine Analogs Produce Atypical Effects without Inducing Inward-Facing Dopamine Transporter Conformations.

Previous structure-activity relationship studies indicate that a series of cocaine analogs, 3ß-aryltropanes with 2ß-diarylmethoxy substituents, selectively bind to the dopamine transporter (DAT) with nanomolar affinities that are 10-fold greater than the affinities of their corresponding 2α-enantiomers. The present study compared these compounds to cocaine with respect to locomotor effects in mice, and assessed their ability to substitute for cocaine (10 mg/kg, i.p.) in rats trained to discriminate cocaine from saline. Despite nanomolar DAT affinity, only the 2ß-Ph2COCH2-3ß-4-Cl-Ph analog fully substituted for cocaine-like discriminative effects. Whereas all of the 2ß compounds increased locomotion, only the 2ß-(4-ClPh)PhCOCH2-3ß-4-Cl-Ph analog had cocaine-like efficacy. None of the 2α-substituted compounds produced either of these cocaine-like effects. To explore the molecular mechanisms of these drugs, their effects on DAT conformation were probed using a cysteine-accessibility assay. Previous reports indicate that cocaine binds with substantially higher affinity to the DAT in its outward (extracellular)- compared with inward-facing conformation, whereas atypical DAT inhibitors, such as benztropine, have greater similarity in affinity to these conformations, and this is postulated to explain their divergent behavioral effects. All of the 2ß- and 2α-substituted compounds tested altered cysteine accessibility of DAT in a manner similar to cocaine. Furthermore, molecular dynamics of in silico inhibitor-DAT complexes suggested that the 2-substituted compounds reach equilibrium in the binding pocket in a cocaine-like fashion. These behavioral, biochemical, and computational results show that aryltropane analogs can bind to the DAT and stabilize outward-facing DAT conformations like cocaine, yet produce effects that differ from those of cocaine.

Differential modulation of methamphetamine-mediated behavioral sensitization by overexpression of Mu opioid receptors in nucleus accumbens and ventral tegmental area.

RATIONALE: Repeated administration of methamphetamine (Meth) induces behavioral sensitization which is characterized by a progressive increase in locomotor response after each injection. Previous studies have shown that Mu opioid receptors (MORs) can regulate Meth-mediated behavioral sensitization. However, the reported interactions are controversial; systemic activation of MORs either enhanced or suppressed Meth sensitization. It is possible that alteration of Meth sensitization after systemic administration of MOR ligands reflects the sum of distinct MOR reactions in multiple brain regions. OBJECTIVES: The purpose of the present study was to examine the actions of MORs on Meth sensitization after regionally selective overexpression of human MOR through an AAV6-based gene delivery system. METHOD: We demonstrated that adeno-associated virus (AAV)-MOR increased MOR immunoreactivity and binding in vitro. AAV-MOR or AAV-green fluorescent protein (GFP) was injected into the nucleus accumbens (NAc) or ventral tegmental area (VTA) of adult mice. Two weeks after viral infection, animals received Meth or saline for five consecutive days. Locomotor behavior and striatal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) level were determined. RESULTS: Repeated administration of Meth progressively increased locomotor activity; this sensitization reaction was attenuated by intra-NAc AAV-MOR microinjections. Infusion of AAV-MOR to VTA enhanced Meth sensitization. AAV-MOR significantly enhanced DA levels in VTA after VTA infection but reduced DOPAC/DA turnover in the NAc after NAc injection. CONCLUSION: Our data suggest a differential modulation of Meth sensitization by overexpression of MOR in NAc and VTA. Regional manipulation of MOR expression through AAV may be a novel approach to control Meth abuse and psychomimetic activity.

Response requirement and increases in accuracy produced by stimulant drugs in a 5-choice serial reaction-time task in rats.

RATIONALE: Increased signal-detection accuracy on the 5-choice serial reaction time (5-CSRT) task has been shown with drugs that are useful clinically in treating attention deficit hyperactivity disorder (ADHD), but these increases are often small and/or unreliable. By reducing the reinforcer frequency, it may be possible to increase the sensitivity of this task to pharmacologically induced improvements in accuracy. METHODS: Rats were trained to respond on the 5-CSRT task on a fixed ratio (FR) 1, FR 3, or FR 10 schedule of reinforcement. Drugs that were and were not expected to enhance performance were then administered before experimental sessions. RESULTS: Significant increases in accuracy of signal detection were not typically obtained under the FR 1 schedule with any drug. However, d-amphetamine, methylphenidate, and nicotine typically increased accuracy under the FR 3 and FR 10 schedules. CONCLUSIONS: Increasing the FR requirement in the 5-CSRT task increases the likelihood of a positive result with clinically effective drugs, and may more closely resemble conditions in children with attention deficits.

Dopamine transporter-dependent and -independent striatal binding of the benztropine analog JHW 007, a cocaine antagonist with low abuse liability.

The benztropine analog N-(n-butyl)-3α-[bis(4'-fluorophenyl)methoxy]-tropane (JHW 007) displays high affinity for the dopamine transporter (DAT), but unlike typical DAT ligands, has relatively low abuse liability and blocks the effects of cocaine, including its self-administration. To determine sites responsible for the cocaine antagonist effects of JHW 007, its in vitro binding was compared with that of methyl (1R,2S,3S,5S)-3-(4-fluorophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (WIN 35428) in rats, mice, and human DAT (hDAT)-transfected cells. A one-site model, with K(d) values of 4.21 (rat) and 8.99 nM (mouse) best fit the [(3)H]WIN 35428 data. [(3)H]JHW 007 binding best fit a two-site model (rat, 7.40/4400 nM; mouse, 8.18/2750 nM), although a one-site fit was observed with hDAT membranes (43.7 nM). Drugs selective for the norepinephrine and serotonin transporters had relatively low affinity in competition with [(3)H]JHW 007 binding, as did drugs selective for other sites identified previously as potential JHW 007 binding sites. The association of [(3)H]WIN 35428 best fit a one-phase model, whereas the association of [(3)H]JHW 007 best fit a two-phase model in all tissues. Because cocaine antagonist effects of JHW 007 have been observed previously soon after injection, its rapid association observed here may contribute to those effects. Multiple [(3)H]JHW 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 35428, the binding of JHW 007 was Na(+)-independent. This feature of JHW 007 has been linked to the conformational status of the DAT, which in turn may contribute to the antagonism of cocaine.

Relationship between conformational changes in the dopamine transporter and cocaine-like subjective effects of uptake inhibitors.

Cocaine exerts its stimulatory effect by inhibiting the dopamine transporter (DAT). However, novel benztropine- and rimcazole-based inhibitors show reduced stimulant effects compared with cocaine, despite higher affinity and selectivity for DAT. To investigate possible mechanisms, we compared the subjective effects of different inhibitors with their molecular mode of interaction at the DAT. We determined how different inhibitors affected accessibility of the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate to an inserted cysteine (I159C), which is accessible when the extracellular transporter gate is open but inaccessible when it is closed. The data indicated that cocaine analogs bind an open conformation, whereas benztropine and rimcazole analogs bind a closed conformation. Next, we investigated the changes in inhibition potency of [(3)H]dopamine uptake of the compounds at a mutant DAT (Y335A) characterized by a global change in the conformational equilibrium. We observed a close relationship between the decrease in potencies of inhibitors at this mutant and cocaine-like responding in rats trained to discriminate cocaine from saline injections. Our data suggest that chemically different DAT inhibitors stabilize distinct transporter conformations and that this in turn affects the cocaine-like subjective effects of these compounds in vivo.

c-Fos facilitates the acquisition and extinction of cocaine-induced persistent changes.

Development of drug addiction involves persistent neurobiological changes. The dopamine D1 receptor is involved in mediating cocaine-induced neuroadaptation, yet the underlying intracellular mechanisms remain unclear. We examined a potential role of the immediate early gene Fos, which is robustly and rapidly induced by cocaine via D1 receptors, in mediating cocaine-induced persistent neurobiological changes by creating and analyzing a mouse in which Fos is primarily disrupted in D1 receptor-expressing neurons in the brain. We show that the expression levels of several transcription factors, neurotransmitter receptors, and intracellular signaling molecules induced by repeated cocaine administration are altered in Fos-deficient brains. Dendritic remodeling of medium spiny neurons induced by repeated exposure to cocaine is blunted in the mutant mice. The mutant mice exhibit attenuated behavioral sensitization after repeated exposure to cocaine and more persistent memory of cocaine-induced conditioned place preference. Our findings indicate that c-Fos produced in D1 receptor-expressing neurons integrates mechanisms to facilitate both the acquisition and extinction of cocaine-induced persistent changes.

Cocaine-induced locomotor activity and cocaine discrimination in dopamine D4 receptor mutant mice.

RATIONALE: Previous studies have found a role for dopamine D(2)-like receptors in many of the behavioral effects of cocaine, including its stimulation of locomotor activity and interoceptive discriminative-stimulus effects. However, given the lack of selectivity of most of the available pharmacological tools among D(2), D(3) and D(4) dopamine receptors, the roles of these specific receptors remain unclear. OBJECTIVES: The roles of specific dopamine D(4) receptors in the behavioral effects of cocaine, including its locomotor stimulant and interoceptive discriminative-stimulus effects were investigated using dopamine D(4) receptor knockout (DA D(4)R KO) and wild-type (WT) mice. METHODS: The mice were trained in daily sessions to discriminate IP injections of saline from cocaine (10 mg/kg). Responses on one of two response keys intermittently produced a food pellet; one response was reinforced in sessions following cocaine injection (10 mg/kg), and the other response was reinforced in sessions following saline injection. Each 20th response produced a food pellet (fixed-ratio, or FR20 schedule of reinforcement). The dose-effects of cocaine and its interaction with the D(2)-like antagonist, raclopride, were assessed. Horizontal locomotor activity was also assessed in each genotype. RESULTS: As previously shown), cocaine was a more potent stimulant of locomotor activity in the DA D(4)R KO mice compared to WT littermate mice. In addition, cocaine was more potent in producing discriminative-stimulus effects in DA D(4)R KO mice (ED(50) value=0.50 mg/kg) compared to their WT littermates (ED(50) value=2.6 mg/kg). Raclopride shifted the cocaine dose-effect curve in both DA D(4)R KO and WT mice, though the shift was greater for the DA D(4)R KO mice. CONCLUSIONS: The present results on the stimulation of activity and interoceptive/subjective effects of cocaine are consistent with the previously reported disregulation of dopamine synthesis in DA D(4)R KO mice, and further suggest a role of the DA D(4)R in vulnerability to stimulant abuse.

Locomotor and discriminative-stimulus effects of cocaine in dopamine D5 receptor knockout mice.

RATIONALE: Dopamine D1-like antagonists block several effects of cocaine, including its locomotor-stimulant and discriminative-stimulus effects. Because these compounds generally lack selectivity among the dopamine D1 and D5 receptors, the specific roles of the subtypes have not been determined. OBJECTIVES: Dopamine D5 receptor knockout (DA D5R KO), heterozygous (HET) and wild-type (WT) mice were used to study the role of D5 dopamine receptors in the effects of cocaine. In addition, effects of the D1-like antagonist, SCH 39166 were also studied to further clarify the roles of D1 and D5 dopamine receptors in the discriminative-stimulus effects of cocaine. METHODS: DA D5R KO, HET and WT mice were treated with cocaine (3-30 mg/kg) or vehicle and their horizontal locomotor activity was assessed. The mice were also trained to discriminate IP injections of saline from cocaine (10 mg/kg) using a two-lever food-reinforcement (FR10) procedure. Doses of cocaine (1.0-10 mg/kg) were administered 5 min before 15-min test-sessions. RESULTS: Cocaine dose-dependently stimulated activity in each genotype, with the highest level of activity induced in the DA D5R WT mice. Both DA D5R KO and HET mice showed reduced levels of horizontal activity compared to WT mice. All three genotypes acquired the discrimination of 10 mg/kg cocaine; doses of 1.0-10.0 mg/kg produced dose-related increases in the number of cocaine-appropriate responses. SCH 39166, at inactive to fully active doses (0.01-0.1 mg/kg) produced predominately saline-appropriate responding. SCH 39166 produced a dose-dependent rightward shift in the cocaine dose-effect curve in all genotypes, with similar apparent affinities. CONCLUSIONS: The present data suggest an involvement of DA D5R in the locomotor stimulant effects of cocaine. In addition, the data indicate that there is little involvement of the DA D5R in the discriminative-stimulus effects of cocaine. In addition, the antagonism data suggest a role of the D1 receptor in the behavioral effects of cocaine.

Cocaine-induced locomotor activity and cocaine discrimination in dopamine D2 receptor mutant mice.

RATIONALE: Dopamine (DA) D2-like antagonists block several effects of cocaine, including its locomotor stimulant and interoceptive discriminative-stimulus effects. Because these compounds generally lack selectivity among the D2-like DA receptors, the specific roles of the subtypes remain unclear. OBJECTIVES: DA D2 receptor knockout (DA D2R KO), heterozygous (HET), and wild-type (WT) mice were used to study the role of D2 DA receptors in the effects of cocaine. Some effects of the relatively selective DA D2-like antagonist raclopride were also studied to further assess the role of D2 receptors. METHODS: DA D2R KO, HET, and WT mice were treated with cocaine (1-10 mg/kg) or vehicle, and their horizontal locomotor activity was assessed. The mice were also trained to discriminate i.p. injections of saline from cocaine (10 mg/kg) using a two-response key, fixed-ratio-20 response, food-reinforcement procedure. A range of doses of cocaine (1.0-17 mg/kg) was administered before 15-min test sessions. RESULTS: Both DA D2R KO and HET mice showed reduced levels of horizontal activity relative to WT mice. Cocaine dose dependently stimulated activity in each genotype, with the highest level of activity induced in the DA D2R WT mice. All three genotypes acquired the discrimination of 10 mg/kg cocaine; tested doses of 1.0-10.0 mg/kg produced dose-related increases in the number of cocaine-appropriate responses. Raclopride, at inactive to fully active doses (0.1-1.0 mg/kg), did not fully substitute for cocaine. Raclopride dose dependently shifted the cocaine dose-effect curve to the right in DA D2R WT and HET mice. However, in DA D2R KO mice, raclopride was inactive as an antagonist. CONCLUSIONS: The present data indicate an involvement of D2 DA receptors in the locomotor-stimulating effects and the interoceptive discriminative-stimulus effects of cocaine in WT subjects. However, the D2 receptor is not necessary for the effects, suggesting redundant dopaminergic mechanisms for the discriminative-stimulus interoceptive effects of cocaine.