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Cocaine is a powerful psychostimulant that is one of the most widely used illicit addictive. The dopamine transporter (DAT) plays a major role in mediating cocaine’s reward effect. Decreases in DAT expression increase rates of drug abuse and vulnerability to comorbid psychiatric disorders. We used the novel DAT transgenic rat model to study the effects of cocaine on locomotor behaviors in adolescent rats, with an emphasis on sex. Female rats showed higher response rates to cocaine at lower acute and chronic doses, highlighting a higher vulnerability and perceived gender effects. In contrast, locomotor responses to an acute high dose of cocaine were more marked and sustained in male DAT heterozygous (HET) adolescents. The results demonstrate the augmented effects of chronic cocaine in HET DAT adolescent female rats. Knockout (KO) DAT led to a level of hyperdopaminergia which caused a marked basal hyperactivity that was unchanged, consistent with a possible ceiling effect. We suggest a role of alpha synuclein (α-syn) and PICK 1 protein expressions to the increased vulnerability in female rats. These proteins showed a lower expression in female HET and KO rats. This study highlights gender differences associated with mutations which affect DAT expression and can increase susceptibility to cocaine abuse in adolescence.

Interest in developing minimal side-effect NMDA receptor antagonists for neurological and psychiatric disorders has been energized by the recent introduction of esketamine into clinical practice for treatment-resistant depression. Structural analogs of dextromethorphan bind with low affinity to the NMDA receptor ion channel, have functional effects in vivo, and generally display a lower propensity for side-effects than that of ketamine and other higher affinity antagonists. As such, the aim of the present study was to determine whether a series of N-substituted-3-alkoxy-substituted dextromethorphan analogs produce their effects in vivo through blockade of NMDA receptors. Compounds were studied against NMDA-induced seizures in rats. Compounds were administered intracerebroventricularly in order mitigate confounds of drug metabolism that arise from systemic administration. Comparison of the anticonvulsant potencies to their affinities for NMDA, σ1, and σ2 binding sites were made in order to help define the receptor population(s) governing NMDA antagonist efficacy. The potencies to block convulsions were positively associated with their affinities to bind to the NMDA receptor ion channel ([(3)H]-TCP binding) (r = 0.71, p < 0.05) but not to σ1 receptors ([(3)H]-SKF 10047 binding) (r = −0.31, p=0.46) or to σ2 receptors ([(3)H]-DTG binding) (p = −0.38, p = 0.36). This is the first report demonstrating that these dextromethorphan analogs are functional NMDA receptor antagonists in vivo. Given their potential therapeutic utility and favorable side-effect profiles, such low affinity NMDA receptor antagonists could be considered for further development in neurological (e.g., anticonvulsant) and psychiatric (e.g., antidepressant) disorders.

Chronic exposure to opioids typically results in adverse consequences. Opioid use disorder (OUD) is a disease of the CNS with behavioral, psychological, neurobiological, and medical manifestations. OUD induces a variety of changes of neurotransmitters/neuropeptides in the nervous system. Existing pharmacotherapy, such as opioid maintenance therapy (OMT) is the mainstay for the treatment of OUD, however, current opioid replacement therapy is far from effective for the majority of patients. Pharmacological therapy for OUD has been challenging for many reasons including debilitating side-effects. Therefore, developing an effective, non-pharmacological approach would be a critical advancement in improving and expanding treatment for OUD. Viral vector mediated gene therapy provides a potential new approach for treating opioid abused patients. Gene therapy can supply targeting gene products directly linked to the mechanisms of OUD to restore neurotransmitter and/or neuropeptides imbalance, and avoid the off-target effects of systemic administration of drugs. The most commonly used viral vectors in rodent studies of treatment of opioid-used disorder are based on recombinant adenovirus (AV), adeno-associated virus (AAV), lentiviral (LV) vectors, and herpes simplex virus (HSV) vectors. In this review, we will focus on the recent progress of viral vector mediated gene therapy in OUD, especially morphine tolerance and withdrawal.

Pharmacological therapies for the treatment of cocaine addiction have had disappointing efficacy, and the lack of recent developments in the clinical care of cocaine-addicted patients indicates a need for novel treatment strategies. Recent studies have shown that vaccination against cocaine to elicit production of antibodies that reduce concentrations of free drug in the blood is a promising method to protect against the effects of cocaine and reduce rates of relapse. However, the poorly immunogenic nature of cocaine remains a major hurdle to active immunization. Therefore, we hypothesized that strategies to increase targeted exposure of cocaine to the immune system may produce a more effective vaccine. To specifically direct an immune response against cocaine, in the present study we have conjugated a cocaine analog to a dendrimer-based nanoparticle carrier with MHC II-binding moieties that previously has been shown to activate antigen-presenting cells necessary for antibody production. This strategy produced a rapid, prolonged, and high affinity anti-cocaine antibody response without the need for an adjuvant. Surprisingly, additional evaluation using multiple adjuvant formulations in two strains of inbred mice found adjuvants were either functionally redundant or deleterious in the vaccination against cocaine using this platform. The use of conditioned place preference in rats after administration of this vaccine provided proof of concept for the ability of this vaccine to diminish cocaine reward. Together these data demonstrate the intrinsic efficacy of an immune-targeting dendrimer-based cocaine vaccine, with a vast potential for design of future vaccines against other poorly immunogenic antigens by substitution of the conjugated cargo.

Smoking commonly is initiated during adolescence: thus, it is important to understand the consequences of early exposure to nicotine. Most studies have focused on male adults; however, in the past 15 years or so, there has been more attention paid to adolescence. More recently, studies have begun to include females. Preclinical laboratory studies show that male adolescents appear to be particularly susceptible to the effects of nicotine and that often the effects are different from adults and from females. Nicotine is more potent as a reward and a stimulant in adolescent males than in females or adults, and the long-term effects are more pronounced than in the other groups. A greater understanding of the differences between adult and adolescent drug responses will aid in the development of appropriate age- and sex-specific treatments for substance abuse.

Women constitute half of all smokers and many studies suggest that adult males and females differ in factors that maintain tobacco smoking, yet there is limited information about sex differences in nicotine reward during adolescence. Limited studies suggest that adolescent male rats self-administer more nicotine than adults, suggesting that drug administration during adolescence leads to different behavioral effects than during adulthood. In the present study, male rats developed a significant conditioned place preference (CPP) to lower doses of nicotine than females, regardless of age. In addition, adolescents were more sensitive than adults. In female rats, adolescents exhibited a CPP of greater magnitude than adult females. In males, the magnitude of the CPP did not differ as a function of age, but adolescents exhibited CPP to lower doses than adults. There also were differences in nicotinic acetylcholinergic receptor binding in nucleus accumbens and caudate putamen in response to nicotine across age and sex. These findings suggest that it is necessary to consider sex- and age-specific effects of drugs such as nicotine when developing strategies for improving smoking cessation treatments.

Studies have shown that many smokers begin using nicotine during adolescence, yet the influence of early nicotine use on the response to other drugs of abuse in adulthood is not fully understood. In the current study, nicotine was administered to adolescent and adult rats for seven days. Thirty days later, cocaine-induced locomotor activity and cocaine self-administration were examined when the rats pretreated as adolescents were adults. Rats exposed to nicotine during early adolescence were sensitized thirty days later to the locomotor-activating effects of cocaine and self-administered a greater number of cocaine infusions than adolescent rats pretreated with vehicle. As a result of this increased intake, the cocaine self-administration dose-response curve was shifted upward indicating an increase in cocaine reinforcement. Rats pretreated with nicotine as adults, however, did not show a difference in locomotor activity or cocaine self-administration thirty days later compared to adult rats pretreated with vehicle. These findings suggest that early exposure to nicotine has long-term consequences on cocaine use. These data further suggest that nicotine use may carry a greater risk during adolescence than adulthood and adolescents who smoke may be particularly vulnerable to stimulant use. This article is part of a Special Issue entitled SI: Adolescent plasticity.

Ecstasy (MDMA) is used predominately by adolescents and young adults. Young MDMA users are more likely than non-users to use other drugs, including cocaine. The response to stimulant drugs can be affected by environmental factors; however, little information exists about the role that housing plays in mediating effects of MDMA in adolescence.The present experiment examined whether social and environmental factors alter effects of MDMA on activity and cocaine reward.Male adolescent rats were housed on PND 23. Isolated rats were housed alone (1 rat/cage) in an impoverished environment with no toys (II) or enriched with toys (IE). Social rats were housed three/cage with (SE3) or without (SI3) toys. Starting on PND 29, 5 mg/kg MDMA or saline was injected and activity was measured for 60 min once daily for five consecutive days. On PND 36-40, cocaine CPP was conducted.Saline vehicle-induced activity of II rats was higher than other groups, and all groups became sensitized to the locomotor-stimulant effects of MDMA. In II rats, maximal CPP was increased after MDMA pre-exposure compared to vehicle. Environmental enrichment blocked this; however, dose-effect curves for cocaine CPP shifted to the left in both IE and SE3 rats. In rats with just social enrichment, there were no effects of MDMA on cocaine CPP.Drug prevention and treatment strategies should take into account different environments in which adolescents live. These findings show that MDMA increases cocaine reward in male adolescents, and social enrichment diminishes, while environmental enrichment enhances this.

Many factors influence the rewarding effects of drugs such as cocaine. The present study was done to determine whether social and environmental factors alter behavior in adolescent male and female rats. On postnatal day (PND) 23, rats were housed in one of several same-sex conditions. Both social (number of rats per cage) and environmental (availability of toys) factors were manipulated. Socially isolated rats were housed alone (1 rat/cage) in an environment that either was impoverished (with no toys; II)) or enriched (with toys; IE). Standard housing for these studies was social and impoverished, which was 2 rats/cage with no toys (SI2). Other rats were housed 2/cage with toys (SE2), or 3/cage with (SE3) or without (SI3) toys. On PND 37, novelty-induced locomotor activity was measured for 30 minutes. On PND 44-46, locomotor activity in response to an injection of 5 mg/kg cocaine was measured for 60 minutes each day. For male rats, only social conditions altered novelty-induced activity. Males housed in groups of three had the most activity, compared to pair-housed and isolated rats. For females, social and environmental enrichment interacted to alter novelty-induced activity. In contrast to males, isolated females had increased activity, compared to group-housed females. Further, isolated females in impoverished environments had more activity than isolated females in enriched environments and group-housed females in impoverished environments. The effect of environmental enrichment on cocaine-stimulated locomotor activity was altered depending upon the number of rats living in a cage for males. For females, only social conditions altered cocaine-stimulated behavior, with activity increasing with the number of rats in the cage, regardless of environmental enrichment. These data show that social and environmental enrichment differentially alter novelty-induced and cocaine-stimulated locomotor activity in adolescent male and female rats.

A series of N1- and N2-substituted 4-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-1,2,3-triazoles were regioselectively synthesized to explore the binding motifs of the diaryl-1,2,3-triazole scaffold at CB1 receptors. The N1 analogs were regioselectively constructed via Click chemistry to furnish analogs with modest CB1 receptor affinity (K i

Marijuana (Cannabis sativa) remains one of the most widely used illegal drugs, with adolescents being particularly vulnerable to its use and abuse. In spite of this, most studies are conducted in adult animals even though the effects might be quite different in adolescents. Additionally, the use of marijuana often precedes the use of other psychoactive drugs including cocaine, especially when marijuana exposure begins during early adolescence. The purpose of this study was to examine the effects of repeated Δ9-tetrahydrocannabinol (THC), the major active ingredient in marijuana, in adolescents compared to adults and to determine its subsequent effects on cocaine-stimulated activity. To this end, adolescent (postnatal day PND 34) and adult (PND 66) rats were administered 3 mg/kg/day THC for 8 days and locomotor activity was measured on days 1, 2, 7 and 8 after dosing. On day 12 (4 days after the last dose of THC), rats were injected with escalating doses of cocaine and behavior was recorded. Results show that THC depressed locomotor activity in adult rats but not in adolescents. However, following a cocaine challenge, adolescents exposed to THC showed increased locomotor responses to cocaine compared to chronic vehicle-injected controls. This was not seen in adults. These results show that the effects of cocaine are enhanced after THC in adolescents, but not adults, and that this might account for the greater transition to cocaine after early, as opposed to later, marijuana use.

The synthesis and structure-activity relationships of 8-substituted-3-[2-(diarylmethoxyethylidenyl)]-8-azabicyclo[3.2.1]octane derivatives were investigated at the dopamine transporter (DAT), the serotonin transporter (SERT) and norepinephrine transporter (NET). The rigid ethylidenyl-8-azabicyclic[3.2.1]octane skeleton imparted modestly stereoselective binding and uptake inhibition at the DAT. Additional structure-activity studies provided a transporter affinity profile that was reminiscent of the structure-activity of GBR 12909. From these studies, the 8-cyclopropylmethyl group has been identified as a unique moiety that imparts high SERT/DAT selectivity. In this study the 8-cyclopropylmethyl derivative 22e (DAT K(i) of 4.0 nM) was among the most potent compounds of the series at the DAT and was the most DAT selective ligand of the series (SERT/DAT: 1060). Similarly, the 8-chlorobenzyl derivative 22g (DAT K(i) of 3.9 nM) was found to be highly selective for the DAT over the NET (NET/DAT: 1358).

A series of 3-arylnortrop-2-enes and 3alpha-arylmethoxy-3beta-arylnortropanes were synthesized and evaluated for binding affinity at monoamine transporters. The 3-(3,4-dichlorophenyl)nortrop-2-ene (6e) exhibited high affinity for the SERT (K(i)=0.3 nM). The 3alpha-arylmethoxy-3beta-arylnortropanes were generally SERT selective with the 3alpha-(3.4-dichlorophenylmethoxy)-3betaphenylnortrop-2-ene (7c) possessing subnanomolar potency (K(i)=0.061 nM). However, 3alpha-(3,4-dichlorophenylmethoxy)-3beta-phenylnortrop-2-ene (7b) exhibited high affinity at all three transporters [(DAT K(i)=22 nM), (SERT K(i)=6 nM) and (NET K(i)=101 nM)].

This study was done to determine whether social and environmental factors alter cocaine reward and proteins implicated in mediating drug reward in rats during early adolescence. On postnatal day (PND) 23, rats were housed under conditions where both social (number of rats per cage) and environmental (availability of toys) factors were manipulated. Socially isolated rats were housed alone impoverished with no toys (II) or enriched with toys (IE). Social rats were housed two rats/cage with no toys (SI2) or with toys (SE2), or three/cage with (SE3) or without (SI3) toys. On PND 43, cocaine conditioned place preference (CPP) sessions began with the post-test done on PND 47. Cocaine CPP was established in response to 5 or 10 mg/kg cocaine in II rats, and CPP was decreased with the addition of cage mates or toys. No CPP was seen to any dose in SI3 or SE3 rats. Enriched housing (SE3) increased dopamine transporter (DAT) protein in the nucleus accumbens compared to II. There also were differential effects of cocaine on tyrosine hydroxylase and DAT depending on housing, with both increased by cocaine in II but not SE3 rats. DARPP-32 was unchanged by housing or cocaine, while phospho-Thr(34)-DARPP-32 was increased by cocaine treatment across conditions. Thus, both social and environmental enrichment decrease cocaine CPP during adolescence and different housing alters proteins that regulate dopaminergic neurotransmission in a manner that may account for the observed differences in cocaine-induced reward.

Human and animal laboratory studies show that adolescents and adults respond differently to drugs and that drug administration during adolescence leads to different behavioral effects than during adulthood. Although there are a number of studies on the effects of cocaine, little is known about the effects of methamphetamine in adolescent vs adult rats. In the present study, sensitivity to the conditioned reward of multiple doses of methamphetamine or cocaine was evaluated in male adolescent (PND 34) and adult (PND 66) rats using a conditioned place preference (CPP) paradigm. In addition, the locomotor-activating effects of methamphetamine were determined across a five-day period of administration. After three days of training with cocaine, both adolescent and adult male rats developed CPP to cocaine, however, the dose-effect curve for cocaine CPP was shifted to the left in adolescent compared to adult rats. In contrast to the development of CPP to cocaine in both groups after three days of conditioning, methamphetamine CPP occurred only in adolescent, and not in adult rats. After five days of training, however, both adolescent and adult rats exhibited identical responses to multiple doses of methamphetamine and a significant CPP was observed in both groups. Daily administration of methamphetamine increased locomotor activity in both adolescent and adult rats, with a greater effect seen in the adults. In neither group, was there evidence of a significant sensitization to the locomotor-activating effects of methamphetamine. These data show that adolescents are more sensitive to psychostimulant reward and thus to the conditioned rewarding properties of cocaine or methamphetamine than adults. A better understanding of this difference may lead to age-specific preventions and treatments for psychostimulant abuse.

Human and animal laboratory studies show that females and males respond differently to drugs and that drug administration during adolescence leads to different behavioral effects than during adulthood. Adult female rats are more sensitive to the behavioral effects of cocaine than adult males, but it is not known if the same effect of sex exists during adolescence. In the present study, sensitivity to the conditioned reward of cocaine was evaluated using a conditioned place preference (CPP) paradigm where adolescent (PND 34) and adult (PND 66) male and female rats were trained and tested for the development of CPP to multiple doses of cocaine. Female rats developed CPP at lower doses than males, regardless of age. In addition, adolescent male and female rats established a CPP at lower doses of cocaine than adult male and female rats, respectively. Thus, both age and sex altered cocaine conditioned reward with the order of sensitivity being adolescent females > adult females > adolescent males > adult males. These data show that adolescents are more sensitive to the conditioned rewarding properties of cocaine than adults and that females respond to lower doses of cocaine compared to males regardless of age.

A series of diarylmethoxymethyltropane-GBR hybrid analogues with all three possible stereochemical orientations at C3 were synthesized and evaluated at dopamine and serotonin transporters. The 3α derivatives were found to be the most potent compounds with the 3α-di(4-fluorophenyl)methoxymethyl-8-(3-phenylpropyl)-8-azabicyclo[3.2.1]octane 15b ( K i = 5 nM) being the most potent compound of the series. The corresponding 3-di(4-fluorophenyl)-methoxymethyl-8-(3-phenylpropyl)-8-azabicyclo[3.2.1]oct-2-ene 12b ( K i = 12 nM) was slightly less potent than the 3α-analogue, while the 3β-di(4-fluorophenyl)methoxymethyl-8-(3-phenylpropyl)-8-azabicyclo[3.2.1]octane 23b ( K i = 78 nM) exhibited only modest affinity for the dopamine transporter. Only the 3α-analogue 15b (SERT/DAT = 48) exhibited higher SERT/DAT selectivity than GBR 12909. These results indicate that the dopamine transporter can tolerate some variability in proximity of the benzhydryl ether to the basic nitrogen atom of the tropane without loss in potency. In addition, the structure–activity data for these tropane-GBR 12909 hybrid analogues support previous findings that the stereochemical and conformational effects imparted by unsaturation at C3 are important for dopamine transporter selectivity over the serotonin transporter.

Nicotine is a popular addictive drug used among the adolescent population, and it has long been questioned whether nicotine use in adolescence may lead to the use of other psychostimulant drugs. It is not fully understood, however, how nicotine alters behavior and brain neurochemistry in the adolescent age cohort and how this may affect subsequent illicit drug use. In the current study, periadolescent and adult male and female rats were treated with nicotine for 7 days. One day or 30 days after this treatment, the effects of amphetamine on locomotor activity were studied. Sensitization to nicotine occurred in periadolescent female and adult male and female rats, but not in periadolescent male rats over the course of the 7-day treatment period. On day 8 (1 day after treatment with nicotine ended) and on day 37 (30 days after treatment with nicotine ended), nicotine-pretreated periadolescent male rats were sensitized to the locomotor-activating effects of amphetamine. The response to amphetamine of periadolescent female and adult male and female rats was unchanged at either time point after nicotine pretreatment. Thus, adolescent males are more sensitive than adults or females to the stimulant effects of amphetamine after exposure to nicotine, and this effect is long-lasting. These data suggest that nicotine use during adolescence may carry a greater risk than during adulthood and that male adolescent smokers may be particularly vulnerable to the risk of stimulant abuse.

Cocaine binds to dopamine (DA), serotonin (5-HT) and norepinephrine (NE) transporters blocking the reuptake of these monoamines into presynaptic terminals. As previously reported, continuous infusion of cocaine for seven days or GBR 12909, a selective dopamine uptake inhibitor, produced significant decreases in prodynorphin (PDYN) gene expression in the hypothalamus. Cocaine also produced a significant increase in PDYN mRNA in the caudate putamen, whereas GBR12909 has no effect and the selective serotonin uptake inhibitor fluoxetine decreases PDYN mRNA in the same brain region. The effect of the selective norepinephrine uptake inhibitor nisoxetine was examined on PDYN gene expression. Nisoxetine or vehicle was infused continuously for 7 days via osmotic minipump into male rats. This treatment produced significant increases in PDYN gene expression in the hypothalamus (183% of control), nucleus accumbens (142% of control) and hippocampus (124% of control) and a significant decrease in the caudate putamen (69% of control). These data suggest that nisoxetine affects PDYN gene expression and support a role for NE in the mechanisms underlying the effects of chronic exposure to psychoactive drugs. Moreover, nisoxetine, as well as fluoxetine, decreases PDYN mRNA in the caudate putamen, in contrast to the up-regulation produced by cocaine. Thus, the inhibition of NE uptake alone cannot account for the cocaine-induced increase of PDYN gene expression. These findings suggest that PDYN gene expression regulation by cocaine in the caudate putamen might be due to a combination of effects on two or three monoamine transporters, or to a mechanism unrelated to transporters inhibition.

Two conformationally constrained tropane derivatives were prepared as rigid nicotinic acetylcholine receptor ligands. A palladium catalyzed intramolecular α-arylation reaction was employed to generate the tricyclic compounds in good yields from N-(bromo-chloropyridylmethyl)-8-azabicyclo[3.2.1]octan-3-ones.

3 beta-Aryltropane analogues wherein the 2-position was substituted with various diarylmethoxyalkyl groups were synthesized and evaluated for binding at the dopamine transporter (DAT), serotonin transporter (SERT), norepinephrine transporter (NET), and muscarinic (M(1)) receptors. The 2 beta-analogues 9a-i generally demonstrated high to moderate binding affinities (K(i) = 34-112 nM) at the DAT with good selectivity over SERT, NET, and M(1) receptors. Alternatively, the 2 alpha-isomers 10a-i were 10-fold less potent at the DAT with poor selectivity over SERT. These SAR studies provide further evidence for the varied binding requirements of structurally diverse tropane-based ligands and support future studies to elucidate DAT binding requirements in relation to cocaine-like behavioral endpoints.

There have been many studies aimed at understanding the role that the dopamine transporter plays in cocaine abuse. Most studies suggest that inhibition of dopamine uptake by cocaine is the primary mechanism by which its behavioral effects are produced. Because of the strong relationship between binding to the dopamine transporter and the behavioral effects of cocaine, the dopamine transporter has on occasion been referred to as the cocaine binding site. Chronic studies using cocaine or selective inhibitors of dopamine, norepinephrine, or serotonin uptake suggest that while a selective dopamine uptake inhibitor can produce sensitization to cocaine, the long-lasting sensitized response to a cocaine challenge observed in cocaine-pretreated rats is due to cocaine's action on a system other than, or in addition to, dopamine. Thus, while dopamine appears to be important for the behavioral effects of cocaine, it appears that neurochemical systems other than dopamine likely play a role in the behavioral effects of chronic cocaine.

The 7315c pituitary tumor cell expresses a homogeneous population of dopamine receptors that are functionally similar to brain dopamine D2 receptors. [3H]-Sulpiride binding to 7315c cell homogenates was specific and saturable, and Ki values for compounds to compete for these sites were highly correlated with values for the same compounds at D2 receptors in brain. Dopamine maximally inhibited approximately 65% of forskolin-stimulated cyclase activity in cell membranes. Some D2 agonists had lower efficacies, suggesting that some compounds are partial agonists at this receptor. Removal of GTP from the assay buffer or pretreatment of the tissue with pertussis toxin abolished the inhibition of adenylyl cyclase by dopamine. Immunodetection of most of the known G alpha subunits revealed that Gi1, Gi2, Gi3, Go, Gq, and Gs are present in the 7315c membrane. Pretreatment with the AS antibody (which recognizes the C-terminal regions of G alpha i and G alpha i2) significantly attenuated the inhibition of adenylyl cyclase activity by dopamine, whereas antibodies to C-terminal regions of the other G alpha subunits had no effect. These findings suggest that the dopamine D2 receptor regulates cyclase inhibition predominantly via Gi1 and/or Gi2 and that the 7315c tumor cells provide a useful model for studying naturally expressed dopamine D2 receptors in the absence of other dopamine receptor subtypes.

Tolerance or sensitization to the locomotor-activating effects of cocaine occurs depending upon the treatment regimen that is used. When cocaine is injected on a daily basis, sensitization occurs, whereas continuously infused cocaine leads to tolerance. Male Sprague-Dawley rats were treated for 7 days with continuous cocaine (50 mg/kg/day) via subcutaneously implanted osmotic minipumps, after which the pumps were removed. Locomotor activity was measured for 1 h each day. Some rats were challenged with an injection of cocaine (7.5, 15 or 30 mg/kg) either 2 or 9 days after pump removal. Two days after the pumps were removed (Day 10), there were no significant differences between cocaine- or saline-treated rats in the amount of locomotor activity produced by the challenge injections. However, cocaine-treated rats challenged with cocaine 9 days after pumps were removed (Day 17) exhibited significant tolerance, as evidenced by a shift downward of the cocaine curve, as compared to saline controls. When the rats were injected again on the next day (Day 18), the activity levels of both groups increased, as compared to the effects observed on Day 17. Thus, although the cocaine-treated rats were still tolerant compared to the saline-treated rats, they were sensitized compared to their previous response to a challenge injection. These findings indicate that tolerance and sensitization to the locomotor-activating effects of cocaine can exist simultaneously, which suggests that they are mediated by separate mechanisms.

Repeated administration of cocaine produces sensitization to its locomotor-activating effects and increases the rate at which cocaine self-administration behavior is acquired. Methylphenidate is administered clinically on a daily basis, predominantly to children and adolescents, for the treatment of attention-deficit hyperactivity disorder (ADHD). It has been demonstrated previously that pretreatment with methylphenidate administered to periadolescent rats decreased the latency to acquisition of cocaine self-administration. Since methylphenidate is often also administered to adults with ADHD, the present study was conducted to determine the effects of prior administration of methylphenidate (5 or 20 mg/kg/day for 9 days) to adult rats on the rate of acquisition for cocaine self-administration (0.25 mg/kg/infusion). The higher dose of methylphenidate significantly decreased the latency for acquisition of this behavior, suggesting that the rats were sensitized to the reinforcing effects of cocaine after treatment with methylphenidate. These findings add to the growing body of evidence suggesting cross-sensitization between the behavioral effects of psychostimulants. Further, insofar as self-administration is a reliable measure of abuse liability, these data suggest that a short-duration pretreatment with a high dose of methylphenidate to adults increases vulnerability to cocaine abuse.

Dopaminergic transmission has been suggested to be a primary mechanism mediating reinforcement, withdrawal and craving associated with psychostimulant addiction. Pyschostimulants attenuate dopamine transporter (DAT) clearance efficiency, resulting in a net increase in synaptic dopamine levels. Re-uptake rate is determined by the number of functional DAT molecules at the membrane surface. Previous in vivo imaging studies in humans and in vitro studies in post-mortem human brain have demonstrated that chronic cocaine abuse results in a neuroadaptive increase in DAT-binding site density in the limbic striatum. Whether this increase in DAT availability represents an increase in the functional activity of the transporter is unknown. Here, we present evidence that DAT function is elevated by chronic cocaine abuse. The effect of increasing post-mortem interval on the functional viability of synaptosomes was modeled in the baboon brain. Baboon brains sampled under conditions similar to human brain autopsies yielded synaptosomal preparations that were viable up to 24 h post-mortem. Dopamine (DA) uptake was elevated twofold in the ventral striatum from cocaine users as compared to age-matched drug-free control subjects. The levels of [3H]DA uptake were not elevated in victims of excited cocaine delirium, who experienced paranoia and marked agitation prior to death. In keeping with the increase in DAT function, [3H]WIN 35,428 binding was increased in the cocaine users, but not in the victims of excited delirium. These results demonstrate that DA uptake function assayed in cryopreserved human brain synaptosomes is a suitable approach for testing hypotheses of the mechanisms underlying human brain disorders and for studying the actions of addictive drugs in man.

A series of eight 2-substituted 3-tolyltropane derivatives were synthesized, and the in vitro and in vivo biological activities as dopamine uptake inhibitors were determined. From the in vitro structure-activity data, it is apparent that a tolyl group in the 2-position, independent of the stereochemical attachment to the tropane ring system, provided compounds (9-12, 14) that exhibit high-affinity binding at the dopamine transporter (DAT). Although a slight stereochemical preference in binding affinity at the DAT was observed for the 2beta-(R)-alcohol 10 over the 2beta-(S)-isomer 11, no significant differences in behavioral effects were observed. Furthermore, despite a relatively low potency of 10 for the inhibition of dopamine uptake compared to its affinity for the DAT, its behavioral profile did not vary significantly from cocaine. These data indicate that a behavioral characterization of compounds is a critical feature of efforts to discover pharmacological treatments for cocaine abuse.

In rodents, saccharin consumption is suppressed when the sweet taste stimulus is paired with moderate doses of cocaine. Several hypotheses have been used to explain the seemingly contradictory effect of decreased consumption of a normally preferred substance following a highly rewarding drug. A common theme across these hypotheses is that saccharin is interpreted as less rewarding after cocaine pairing. We considered the alternative possibility that suppression is caused not by a change in reward circuitry, but rather by a change in taste detection, for instance by altering the afferent taste response and decreasing sensitivity to sweet taste stimuli. To evaluate this possibility, we measured saccharin taste sensitivity of mice before and after a standard cocaine-pairing paradigm. We measured taste sensitivity using a brief-access lickometer equipped with multiple concentrations of saccharin solution and established concentration-response curves before and after saccharin-cocaine pairing. Our results indicate that the EC50 for saccharin was unaltered following pairing. Instead, the avidity of licking saccharin, an indicator of motivation, was depressed. Latency to first-lick, a negative indicator of motivation, was also dramatically increased. Thus, our findings are consistent with the interpretation that saccharin-cocaine pairing results in devaluing of the sweet taste reward.

kappa-Opioid receptor agonists have been suggested as treatments for cocaine addiction based on studies showing that they block cocaine-related behaviors. To determine the effects of kappa-opioid receptor agonists on long-term behavioral effects associated with cocaine and the neurochemical bases underlying these effects, rats were treated with the selective kappa-opioid receptor agonist U-69593 ((+)(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidinyl)-1 oxaspiro[4.5]dec-8-yl]-benzeneacetamide) alone or in combination with cocaine and locomotor activity was measured daily. In addition, dopamine transporter and dopamine receptor densities were measured using autoradiographic techniques, and tyrosine hydroxylase was measured using immunoautoradiographic techniques. Treatment with U-69593 with or without cocaine decreased locomotor activity. When challenged with cocaine after a 5-day treatment period, the effects of cocaine were markedly reduced in rats initially treated with U-69593 compared to vehicle. When U-69593 was administered five times with 3-day intervals, it alone had no effect on locomotor activity but still reduced activity associated with a cocaine injection. After five daily injections, U-69593 decreased dopamine transporter and dopamine D(2) receptor densities and increased tyrosine hydroxylase levels. These changes were not seen after the 3-day interval regimen, even though cocaine-induced activity was greatly reduced. These findings show that the effects associated with daily U-69593 treatment are attenuated if the drug is administered with a greater interval, while maintaining a blockade of cocaine-induced activity. In addition, U-69593 can block cocaine-induced locomotor effects without major perturbation of the dopamine system.

Partial opioid agonists can produce their actions at opioid as well as some non-opioid sites. Although the receptor systems underlying these non-opioid effects are not completely clear, recent studies indicate the possible involvement of activity at the dopamine uptake site. One purpose of the present investigation was to examine the ability of selected partial opioid agonists (dezocine, meperidine and [+]-propoxyphene) with non-opioid actions to produce cocaine-like stimulus effects. Because non-opioid effects can be apparent under conditions in which opioid-mediated effects are blocked or at doses that markedly decrease responding, these opioids were also examined in combination with the opioid antagonist naltrexone. A second purpose was to determine the ability of these opioids to inhibit [3H]dopamine uptake in rat caudate putamen. Cocaine and the direct-acting dopamine agonist (-)-quinpirole, but not (+)-propoxyphene, butorphanol, morphine, U50,488 and pentobarbital, substituted completely for the cocaine stimulus. Dezocine substituted for the cocaine stimulus in the majority of the rats tested only when administered in combination with naltrexone. Meperidine also substituted for the cocaine stimulus in the majority of the rats tested, although this pattern of substitution was not consistently altered by naltrexone. Dezocine and meperidine inhibited [3H]dopamine uptake in a manner consistent with that produced by cocaine. The results suggest that dezocine and meperidine can produce cocaine-like stimulus effects and that these effects are likely mediated by activity at the dopamine uptake site.

To better understand the influence of kappa-opioid agonists on the effects of cocaine, rats were treated with daily injections of the selective kappa-opioid agonist U-69593 or bremazocine. In combination with 10 mg/kg cocaine, both compounds, at a dose of 0.32 mg/kg, greatly diminished locomotor activity, and these effects were maintained over a period of 5 days. In addition, the response to a challenge injection of 10 mg/kg cocaine several days after the end of kappa-Opioid agonist treatment with or without cocaine was markedly reduced. When naltrexone was given in combination with U-69593, it blocked the reduction in cocaine-induced locomotor activity after U-69593 treatment alone. However, a single injection of either kappa-opioid agonist alone had no effect on cocaine-induced locomotion several days later (i.e. no long-term effects), suggesting that multiple injections of the kappa-opioid agonist are needed to reduce the locomotor activating effects of cocaine other than acutely. In addition, treatment with the kappa-opioid agonist U-69593 (0.32 mg/kg) over a 5-day period decreased dopamine transporter densities in the caudate putamen, and this was also blocked by co-administration of naltrexone.

The effect of the selective dopamine uptake inhibitor 1-[2-[bis(4-flourophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine dihydrochloride (GBR 12909) was examined on prodynorphin gene expression. GBR 12909 or vehicle was continuously infused for 7 days via osmotic minipump, or injected daily into male rats. Both continuous infusions and daily injections of GBR 12909 produced significant decreases in prodynorphin expression in the hypothalamus (37% and 31% decreases, respectively). There were no significant changes in the caudate putamen, hippocampus or nucleus accumbens. One injection of GBR 12909 had no effects on prodynorphin expression in any of the brain regions studied, suggesting that the effect in the hypothalamus is not an acute effect. As previously reported for other treatment regimens, continuous infusion of cocaine produced a 35% significant decrease in the hypothalamus, consistent with the effects of GBR 12909. In contrast to GBR 12909, however, cocaine also produced a significant increase in prodynorphin expression in the caudate putamen. Thus, chronic inhibition of dopamine uptake can regulate prodynorphin expression in the hypothalamus. In contrast, the increase in the caudate putamen following cocaine administration may not be related to the inhibition of dopamine uptake, since it was not produced by a selective dopamine uptake inhibitor. These findings suggest that regulation of prodynorphin gene expression by cocaine in the caudate putamen may be mediated by the inhibition of norepinephrine or serotonin uptake, by a combination of effects on two or three monoamine transporters, or by a mechanism unrelated to transporter inhibition.

Rationale: The discriminative-stimulus effects of cocaine have been reported to be mediated by indirect agonist actions initiated by the blockade of dopamine uptake, and the potencies of drugs that have discriminative-stimulus effects like cocaine are directly related to their dopamine transporter binding affinities. The binding to the dopamine transporter by cocaine and many of its analogs has been reported to fit better using a two-site model than a one-site model. Objectives: The present study examined the relationship among binding affinities of dopamine uptake inhibitors at these two sites and their potencies to produce discriminative-stimulus effects. Methods: The inhibition constants (K i values) were derived for unlabeled dopamine uptake inhibitors for displacement of [3H]WIN 35,428 from rat caudate putamen membranes. These K i values were related to the ED50 values obtained in rats trained to discriminate 10 mg/kg cocaine from saline injections under a fixed-ratio 20 schedule of food reinforcement. Results: Among the dopamine uptake inhibitors studied, the binding data for eight compounds (WIN 35,428, nomifensine, WIN 35,981, WIN 35,065-2, methylphenidate, cocaine, cocaethylene, and bupropion) were better fit by a two-site model than a one-site model. The data for the remaining eleven compounds (RTI-31, RTI-55, RTI-121, RTI-32, LU19-005, BTCP, GBR12909, GBR12935, mazindol, LU17-133, and EXP561) were better fit by a one-site model. Of the drugs that were fit best by a two-site model, there was a higher correlation among the K i values for the high-affinity site and the ED50 values (R2=0.655; P=0.015) than there was for the low-affinity site (R2=0.543; P=0.037). Of the remaining drugs, there was a high correlation among the K i values and the ED50 values for the discriminative-stimulus effects (R2=0.523; P=0.012). Conclusions: These data suggest that the discriminative-stimulus effects of cocaine are more closely related to actions mediated by high-affinity binding to the dopamine transporter than they are to actions mediated by the low-affinity site. The further assessment of the respective contributions of high- and low-affinity binding to the behavioral effects of cocaine will be greatly enhanced with the development of pharmacological tools that have a high degree of selectivity for one of these components.

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 of1.3 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 to6 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.

A series of phenyl ring-substituted analogues of 3alpha-(diphenylmethoxy)tropane (benztropine) has been prepared as novel probes for the dopamine transporter. Cross-validated comparative molecular field analysis (CoMFA) models of the binding domain on the dopamine transporter were constructed using 37 geometry-optimized structures of these compounds and their corresponding binding affinities (K(i) values) for the displacement of [(3)H]WIN 35,428 or potency of [(3)H]dopamine uptake inhibition (IC(50) values) in rat caudate putamen tissue. The most predictive model (q(2) = 0.78) correlated the steric component of CoMFA to the dependent variable of [(3)H]WIN 35,428 binding affinities. A novel series of seven phenyl ring-substituted analogues of 3alpha-(diphenylmethoxy)tropane was prepared, and our best molecular model was used to accurately predict their binding affinities. This study is the first to provide a CoMFA model for this class of dopamine uptake inhibitors. This model represents an advancement in the design of novel dopamine transporter ligands, based on 3alpha-(diphenylmethoxy)tropane, and further substantiates structure-activity relationships that have previously been proposed for this class of compounds. This CoMFA model can now be used to predict the binding affinities of novel 3alpha-(diphenylmethoxy)tropane analogues at the dopamine transporter and will be useful in the design of molecular probes within this class of dopamine uptake inhibitors.

Chronic continuous cocaine treatment produces a unique pattern of locomotor activation over time. An initial, progressive increase in locomotion is indicative of sensitization. Unlike intermittent cocaine, this increase is subsequently reversed during the continuous exposure, and activity returns to pre-sensitization levels within days. To study the pharmacological mechanisms that underlie this phenomenon, osmotic minipumps containing cocaine or selective uptake inhibitors of dopamine (GBR 12909 or RTI-117), serotonin (fluoxetine), or norepinephrine (nisoxetine) were implanted into rats. Locomotor activity was measured for 1 h each day, beginning 4 h after pumps were implanted. In the cocaine group, activity was significantly elevated on the first day, peaked between the second and third days, then decreased to a plateau which remained significantly above control levels through 14 days. Peak activity in the GBR 12909 and RTI-117 animals occurred on the first day, followed by a significant decrease 24-48 h later, but not complete tolerance. Neither fluoxetine nor nisoxetine altered locomotor activity. The selective dopamine uptake inhibitors produced some of the effects of cocaine. The possibilities that cocaine interacts with the dopamine transporter in a qualitatively different manner from that of these selective dopamine uptake inhibitors, or that other monoamine systems are involved, are discussed.

U-69593, the selective K-opioid agonist, was repeatedly administered in single daily injections (0.32 mg/kg) to male, Sprague-Dawley rats. Two or ten days later, the rats were euthanized and dopamine D1 and D2 receptors were measured using (3H]SCH 23390 or [3H]sulpiride, respectively, in caudate putamen and nucleus accumbens. Two days after the last of three injections, dopamine D2 receptors in the caudate putamen were decreased by approximately 40%, with no change in D1 receptors. Dopamine D2 receptor number had returned to normal by 10 days posttreatment. In contrast, in the nucleus accumbens there was a small, nonsignificant decrease in dopamine D2 receptors 2 days after treatment, but a large increase (65%) after 10 days. In agreement with the changes in D2 receptors, there was a significant downward shift in the locomotor activity curve for the D2 agonist quinpirole after a 2-day withdrawal. There were no differences in either the total amount of dopamine taken up or in the IC50 for cocaine to inhibit dopamine uptake following this treatment, suggesting that the dopamine transporter and presynaptic terminals were intact. The results of these studies demonstrate that repeated administration of a selective K-opioid agonist induces long-term alterations in dopamine D2 receptors. Furthermore, the finding that these changes in receptor number require both repeated injections and a withdrawal time greater than 1 day suggests that these alterations are compensatory in nature.

Some sigma receptor ligands have been shown to bind with low affinity to the dopamine transporter and to inhibit [3H]dopamine uptake. It has not previously been shown whether any of these compounds influence release of dopamine via facilitated exchange diffusion. To further examine the nature of the interaction between sigma receptor ligands and the dopamine transporter, the effects of sigma receptor ligands on amphetamine-stimulated [3H]dopamine release were examined in slices prepared from rat caudate putamen. In the absence of exogenous Ca2+, both (+)-pentazocine and (-)-pentazocine potentiated amphetamine-stimulated [3H]dopamine release at concentrations consistent with their affinities for sigma2 receptors. In contrast, BD737 (1S.2R-(-)-cis-N-¿2-(3,4-dichlorophenyl)ethyl¿-N-methyl-2-(1-pyrrolidiny l)cyclohexylamine), a sigma1 receptor agonist, had no effect on amphetamine-stimulated release. Neither isomer of pentazocine alone had any effect on basal [3H]dopamine release under these conditions. Three antagonists at sigma receptors, one of which is non-selective for subtypes, and two of which are sigma2-selective, all blocked the enhancement of stimulated release produced by (+)-pentazocine. Enhancement of stimulated release by (-)-pentazocine was similarly blocked by sigma2 receptor antagonists. Our data support the contention that it is possible to regulate transporter-mediated events with drugs that act at a subpopulation of sigma receptors pharmacologically identified as the sigma2 subtype.

A novel photoaffinity label for the dopamine transporter (DAT) based on N-substituted 3α-[bis(4′-fluorophenyl)methoxy]tropane has been synthesized in five steps and has been characterized. Preliminary binding studies indicated this ligand bound irreversibly to the dopamine transporter. Preparation of the 125I analog and its photoactivation in the presence of membrane bound DAT demonstrated it covalently binds to the DAT.

The chronic continuous infusion of cocaine produces partial behavioral tolerance to cocaine and tolerance to the inhibition of dopamine uptake by cocaine, without changing dopamine transporter binding. In order to examine more closely the dopaminergic contribution to this effect, the selective dopamine uptake inhibitor GBR 12909 (30mg/kg/day), cocaine (50mg/kg/day), or vehicle, were continuously infused via osmotic minipump, and their effects on the dopamine transporter examined. Drug and vehicle pumps were implanted into male Sprague-Dawley rats and removed after seven days. [3H]WIN 35,428 binding and [3H]dopamine uptake were measured in caudate putamen and nucleus accumbens at varying intervals after pump removal. The B max for [3H]WIN 35,428 binding was decreased by approximately 75% in the caudate putamen and by 40% in the nucleus accumbens of GBR 12909-treated rats both 1 and 4 days after pump removal, and was still significantly decreased after 10 days, but had returned to normal by 20 days post-treatment. In contrast, cocaine did not significantly alter [3H]WIN 35,428 binding. GBR 12909 produced both tolerance to the inhibition of [3H]dopamine uptake by cocaine, and a decrease in total uptake of dopamine, in the caudate putamen, with no change in the nucleus accumbens. The persistent reduction of [3H]WIN 35,428 binding following continuous GBR 12909 does not appear to result from residual drug binding. These findings suggest that GBR 12909 and cocaine may bind to and regulate the dopamine transporter in different ways.

Both in vitro binding studies and studies of dopamine uptake have indicated that there is a heterogeneity of action of cocaine and cocaine analogs. Both high- and low-affinity binding sites have been identified. Some drugs that bind to the dopamine transporter show both high- and low-affinity components whereas others do not. Behavioral studies have indicated that the high-affinity component appears to be the one most directly involved in the actions of cocaine related to abuse. These conclusions are based on correlations of affinities and psychomotor stimulant effects. In addition, tolerance to the psychomotor stimulant effects of cocaine occurs with a concomitant change in only the high-affinity component for dopamine uptake. Certain dopamine uptake inhibitors may have only actions mediated by the low-affinity component. These drugs bind to the dopamine transporter and inhibit dopamine uptake; however, they do not have behavioral effects like those of cocaine. This finding is a critical point of inquiry for the dopamine hypothesis because, based on the neurochemical data, these drugs should have behavioral actions like those of cocaine. In contrast, some of these drugs antagonize the behavioral effects of cocaine, suggesting that the low-affinity site somehow modulates the actions mediated by the high-affinity site. Recently, some benztropine analogs have been discovered that bind to the dopamine transporter and inhibit dopamine uptake monophasically but have behavioral effects that are dissimilar to those of cocaine. These compounds may prove useful in determining the behavioral significance of heterogeneity of actions at the dopamine transporter. Further, these studies may provide leads to novel therapeutics for the treatment of cocaine abuse.

A series of 3-aryl-3-arylmethoxy-azetidines were synthesized and evaluated for binding affinities at dopamine and serotonin transporters. The 3-aryl-3-arylmethoxyazetidines were generally SERT selective with the dichloro substituted congener 7c (Ki = 1.0 nM) and the tetrachloro substituted derivative 7i (Ki = 1.3 nM) possessing low nanomolar affinity for the SERT. The 3-(3,4-dichlorophenyl-3-phenylmethoxyazetidine (7g) exhibited moderate affinity at both DAT and SERT transporters and suggests that substitution of the aryl rings can be used to tune the mononamine transporter affinity.

Cocaine alters opioid receptor densities in rat brain. To investigate the functional consequences of such opioid receptor changes, adenylyl cyclase activity was measured in rat nucleus accumbens and caudate putamen following continuous cocaine administration (50 mg/kg/day, 7 days). In the nucleus accumbens chronic cocaine led to an increase in both the number of mu-opioid receptors and the maximal inhibition of adenylyl cyclase activity by DAMGO ([D-Ala2,N-methyl-Phe4,Glyol]enkephalin). There was no effect on inhibition of adenylyl cyclase activity by DPDPE ([D-Pen2,D-Pen5]enkephalin). There were no changes in the caudate putamen. Thus, continuous cocaine administration for 7 days results in a selective increase in mu-opioid receptor-mediated effector function in the nucleus accumbens.

A series of 9-methyl-3 beta-phenyl-2-substituted-9-azabicyclo[3.3.1]nonane derivatives were synthesized and evaluated as cocaine-binding site ligands at the dopamine transporter (DAT). The conformation of the bicyclic structures and the stereochemistry of the substituents were determined by NMR and X-ray crystallography. The in vitro binding affinity (Ki) of the 9-azabicyclo[3.3.1]nonane derivatives was measured in rat caudate-putamen tissue, and they were found to be 100-fold (Ki = 2-14 microM) less potent than cocaine and other tropane analogs. From these results it is evident that the cocaine-binding site at the DAT is very sensitive to structural modifications of the unsubstituted methylene bridge [C(6)-C(7)] of cocaine and cocaine-like compounds.

The transition metal complexes [eta(6)- (2beta-carbomethoxy-3beta-phenyltropane)]tricarbonylchromium (3) and [eta(6)-(2beta-carbomethoxy-3beta-phenyltropane)] [eta(5)-(pentamethylcyclopentadienyl)]ruthenium(II)triflate (4) were synthesized from 2beta-carbomethoxy-3beta-phenyltropane (2, WIN 35,065) to further elucidate the influence of substituents on the 3beta-aryl on the affinity of the ligand for cocaine-binding sites at the dopamine transporter. The compounds were tested for their ability to displace bound [(3)H]WIN 35,428 (5) from rat caudate putamen tissue and for their ability to inhibit [(3)H]dopamine uptake. The binding affinity for 3 was 2-fold greater than those observed for cocaine (1) and 2, while the binding affinity for 4 was found to be 100-fold less than those of 1 and 2. In addition, 3 was equipotent with 1 and 2 in [(3)H]dopamine uptake inhibition studies, while 4 was 10-fold less potent. The potencies of the complexes 3 and 4 correlated well with the structure-activity relationships of other 2beta-carbomethoxy-3beta-aryltropane derivatives. These data further support a pharmacophore model in which the region occupied by the aryl ring is a lipophilic pocket with electropositive character.

(±)-(N-Alkylamino)benzazepine analogs were prepared as novel dopamine D 1 receptor antagonists to further elucidate the role of these receptor subtypes in the pharmacology and toxicology of cocaine. In the first series of compounds, (±)-7-chloro-8-hydroxy-3-[6-(N,N-dimethylamino)-hexyl]-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (15) showed the highest affinity (K i = 49.3 nM) and subtype-selectivity for dopamine D 1 over dopamine D 2 , 5-HT 2a , and 5-HT 2c receptors. Compounds 7a {(i)-7-Chloro-8-hydroxy-3-[4-(N,N-dimethylamino)butyl]-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine}, 11 {(±)-7-chloro-8-hydroxy-3-[6-[(N,N-dimethylamino)hexyl]-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-cyanoborane), and 15 were moderately potent dopamine D 1 receptor antagonists as evidenced by their ability to block dopamine-stimulated adenylyl cyclase activity in rat caudate (predicted K i values = 60, 34, and 21 nM, respectively). Compound 7a appears to be unique in that, despite its relatively potent inhibition of dopamine stimulated adenylyl cyclase, it demonstrated relatively weak binding affinity at the dopamine D 1 receptors (K i = 811 nM). Unlike previously reported N-alkylbenzazepines, where a significant loss in dopamine D 1 receptor binding affinity was observed when successive increases in the alkyl side chain size at the benzazepine nitrogen were made, several of these novel N-alkylamino analogs demonstrated high-affinity binding with an optimal chain length of six carbons. This initial series of compounds appears to be identifying another binding domain on the dopamine D 1 receptor protein that has not previously been characterized and that accepts an amino function. Further, these compounds may serve as templates for the design of peripherally active dopamine D 1 receptor antagonists

A series of 4'-substituted and 4',4"-disubstituted 3 alpha-(diphenylmethoxy)tropane analogs were prepared as novel probes for the dopamine transporter. These compounds were evaluated in radiolabeled binding assays for the dopamine, norepinephrine, and serotonin transporters. All of these compounds monophasically displaced [3H]WIN 35,428 binding in rat caudate putamen with Ki values ranging from 11.8 to 2000 nM. The most potent compound in this series was 4',4"-difluoro 3 alpha-(diphenylmethoxy)tropane 7c with a Ki = 11.8 nM. All of the compounds inhibited dopamine uptake in rat caudate putamen (IC50 = 24-4456 nM) which correlated significantly (r = 0.907; p > 0.0001) with binding affinities at the dopamine transporter. None of the compounds demonstrated high-affinity binding at the norepinephrine (Ki > 4800 nM) or serotonin (Ki > 690 nM) transporters. Therefore, the most potent dopamine uptake inhibitors in this series were highly selective for the dopamine transporter. Preliminary behavioral studies of several of these analogs (7a-e) suggested that the compounds did not display a cocaine-like behavioral profile, despite their ability to inhibit dopamine uptake. The present data coupled with the observed differences from cocaine in structure-activity relationships suggested that the 3 alpha-(diphenylmethoxy)tropane analogs may be interacting at a different active site than cocaine on the dopamine transporter and that an additional binding domain might be exploited for the identification of potential therapeutics for the treatment of cocaine abuse.

Carbetapentane (1, 2-[2-(diethylamino)ethoxy]ethyl 1-phenyl-1-cyclopentanecarboxylate) binds with high affinity to sigma sites and is a potent antitussive, anticonvulsant, and spasmolytic agent. However, carbetapentane interacts at muscarinic binding sites as well, and it is not clear whether either of these receptor systems is involved in the mechanism(s) of action(s) of this drug. In an attempt to determine whether these psychoactivities can be attributed to interaction at sigma sites, a series of carbetapentane analogs were prepared. Phenyl ring substitution; contraction, expansion, and replacement with a methyl group of the cyclopentyl ring; replacement of the carboxylate function with an amide, methyl ether, and methylamine; and replacement of the N,N-diethyl substituent with a morpholino or piperidino moiety were investigated. All of these novel analogs were evaluated for binding to sigma 1 and sigma 2 sites, and comparison of binding at muscarinic m1 and m2 and PCP (1-(1-phenylcyclohexyl)piperidine) receptors was performed. All of the compounds were selective for sigma 1 over sigma 2 sites, with the three most selective analogs being compounds 34 (65-fold), 35 (78-fold), and 39 (51-fold). None of the compounds were active at PCP sites, and chemical modification including (1) replacing the ester function, (2) replacing the cyclopentyl ring with a smaller ring system (cyclopropyl) or a methyl group, and (3) replacing the diethylamino moiety with a morpholino group resulted in > 220-fold selectivity over muscarinic receptor binding. Therefore, several of these novel compounds are potent, sigma 1-selective ligands which can now be investigated as potential antitussive, anticonvulsant, and antiischemic agents. These studies may reveal whether sigma 1 sites play a role in the pharmacological actions of these drugs.