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.
A series of N-substituted 3 alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues has been prepared that function as dopamine uptake inhibitors. The N-methylated analogue of this series had a significantly higher affinity for the dopamine transporter than the parent compound, N-methyl-3 alpha- (diphenylmethoxy)tropane (benztropine, Cogentin). Yet like the parent compound, it retained high affinity for muscarinic receptors. A series of N-substituted compounds were prepared from nor-3 alpha-[bis(4'-fluorophenyl)methoxy]tropane via acylation followed by hydride reduction of the amide or by direct alkylation. All compounds containing a basic tropane nitrogen displaced [3H]WIN 35,428 at the dopamine transporter (Ki range = 8.5-634 nM) and blocked dopamine uptake (IC50 range = 10-371 nM) in rat caudate putamen, whereas ligands with a nonbasic nitrogen were virtually inactive. None of the compounds demonstrated high binding affinity at norepinephrine or serotonin transporters. Importantly, a separation of binding affinities for the dopamine transporter versus muscarinic m1 receptors was achieved by substitution of the N-methyl group with other N-alkyl or arylalkyl substituents (eg. n-butyl, allyl, benzyl, 3-phenylpropyl, etc.). Additionally, the most potent and selective analogue in this series at the dopamine transporter, N-(4"-phenyl-n-butyl)-3 alpha-[bis(4'-fluorophenyl)methoxy]tropane analogue failed to substitute for cocaine in rats trained to discriminate cocaine from saline. Potentially, new leads toward the development of a pharmacotherapeutic for cocaine abuse and other disorders affecting the dopamine transporter may be discovered.
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 postmortem 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 [ 3 H]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, [ 3 H]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.
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.
SummaryHuman 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.
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