High-affinity [3H]GBR 12783 binding to a specific site associated with the neuronal dopamine uptake complex in the central nervous system

We labelled the neuronal dopamine uptake system by using the potent dopamine uptake inhibitor GBR 12783 in its tritiated form (18.3 Ci/mmol). The binding of [3H]GBR 12783 to rat striatal membranes was saturable and specific with a Kd of 1.6 nM and a Bmax of 10.3 pmol X mg protein-1 as determined by Scatchard analysis. [3H]GBR 12783 binding to rat striatal membranes was inhibited by dopamine uptake inhibitors with IC50 highly correlated with their IC50 for inhibiting [3H]dopamine uptake by a rat striatal synaptosomal preparation. The rank order of potency was the following: GBR 12783 greater than amfonelic acid greater than mazindol greater than pyrovalerone greater than nomifensine greater than benztropine greater than amineptine greater than methylphenidate greater than cocaine. Substrates of dopamine uptake competed with [3H]GBR 12783 binding at concentrations higher than those at which they inhibited [3H]dopamine uptake. In rats with a unilateral section of the medial forebrain bundle, the decrease in [3H]GBR 12783 binding to membranes prepared from the ipsilateral striatum was equal to the decrease in [3H]dopamine uptake by a synaptosomal preparation obtained from the same striatum. [3H]GBR 12783 bound in a sodium-dependent manner to membranes prepared from striatum, nucleus accumbens and tuberculum olfactorium. GBR 12783 displayed an approximately 150-fold lower affinity for the cortical norepinephrine uptake system labelled with [3H]desipramine than for the dopamine transport complex labelled with [3H]GBR 12783. [3H]GBR 12783 appears an attractive tool for the selective characterization of the dopamine uptake system in vitro.     

3H]GBR-12935 binding sites in human striatal membranes: binding characteristics and changes in parkinsonians and schizophrenics

The binding of the diphenyl-substituted piperazine derivative, [3H]GBR-12935, a selective dopamine uptake inhibitor, to the post-mortem human putamen was studied. Inhibition curves by dopamine uptake inhibitors suggested the existence of two populations of [3H]GBR-12935 binding sites: one is potently inhibited by mazindol and/or nomifensine, and the second binding site is benztropine- and/or GBR 12909-sensitive. In the human putamen, [3H]GBR-12935 labeled both these two distinct binding sites. The [3H]GBR-12935 binding displaced by mazindol was enriched in the mouse and rat striatum, but not in the cultured mouse neuroblastoma cell N1E-115. The mazindol-sensitive [3H]GBR-12935 binding site increased in the presence of sodium and markedly decreased in the putamen from parkinsonians (45% of controls). On the other hand, the [3H]GBR-12935 binding displaced by benztropine showed no sodium-dependent increase and was not decreased in the putamen from parkinsonians. In the putamen from schizophrenics, the [3H]GBR-12935 binding did not significantly change in the density, while that displaced by mazindol tended to increase. It is concluded that in the human putamen, [3H]GBR-12935 binds to two distinct sites. One site is partially sodium-dependent and appears to be associated with a high-affinity dopamine uptake system on dopaminergic nerve terminals. The second binding site shows no sodium-dependency and may be associated with a nondopaminergic and/or extraneuronal DA uptake system.     

3H]GBR 12935 binding in vivo in mouse brain: labelling of a piperazine acceptor site

The binding of the selective dopamine uptake inhibitor [3H]GBR 12935 was studied in vivo in mouse brain. The binding was reversible with t1/2 = 80 min. The localisation of [3H]GBR 12935 binding and of dopaminergic receptors did not overlap. The binding of [3H]GBR 12935 was distributed almost uniformly throughout the brain. Also, the in vitro inhibition of dopamine uptake and the inhibition of in vivo [3H]GBR 12935 binding did not correlate when a series of relevant reference compounds was used. The potencies of various dopamine uptake inhibitors to induce stereotyped behavior did not correspond to the inhibitory potencies in the [3H]GBR 12935 binding assay. In conclusion, [3H]GBR 12935 labels in vivo a site which is different from the dopamine uptake complex. We have recently obtained results for the in vitro binding of [3H]GBR 12935 that indicate that this site could be a piperazine acceptor site.     

Interaction of [3H]GBR 12935 and GBR 12909 with the dopamine uptake complex in nucleus accumbens

Although some behavioral effects of cocaine are hypothesized to be due to blockade of dopamine uptake in nucleus accumbens, it has been reported that in nucleus accumbens there are no specific cocaine binding sites and that cocaine is a weak inhibitor of dopamine uptake. [3H]GBR 12935 and an unlabelled analog, GBR 12909, are ligands that bind with great affinity and specificity to a site on dopamine uptake complex in striatum. We therefore investigated the interaction of these GBR compounds with the dopamine uptake complex in nucleus accumbens. We found specific high affinity [3H]GBR 12935 binding and a significant correlation between displacement of [3H]GBR 12935 binding by a series of compounds in striatum and nucleus accumbens. GBR 12909 inhibited dopamine uptake with equal potency in nucleus accumbens and striatum. Thus, there appear to be some aspects of the dopamine uptake complex in nucleus accumbens and striatum that are similar.     

3H]GBR 12935 binding to dopamine uptake sites: subcellular localization and reduction in Parkinson's disease and progressive supranuclear palsy

[3H]GBR 12935 bound with high affinity to dopamine uptake sites in rat striatum where a close parallelism was observed between the subcellular localization profiles for [3H]dopamine uptake and [3H]GBR 12935 specific binding. Using the same ligand, we characterized the dopamine uptake sites in human striatum: the mean KD value was 3.2 nM and the specific binding was inhibited by several dopamine uptake blockers but with slightly lower affinities than those observed in the rat. The subcellular localization profile revealed a synaptosomal enrichment of the specific binding in human striatum. [3H]GBR 12935 binding was decreased in the putamen and caudate nucleus of subjects with Parkinson's disease (33 and 46% of control values, respectively) and progressive supranuclear palsy (38 and 57% of control values, respectively). It is very unlikely that the remaining binding sites in both diseases correspond to piperazine acceptor sites that are not involved in dopamine uptake. However, we cannot exclude the possibility that some of these remaining dopamine transporter sites are not functional, since the reduction in [3H]GBR 12935 specific binding was less marked than the decrease in the dopamine content of the same areas.     

Radiolabeling of dopamine uptake sites in mouse striatum: comparison of binding sites for cocaine,mazindol, and GBR 12935

Summary This study addressed the possibility of a unique binding interaction between cocaine and the dopamine transporter as compared with other blockers of dopamine uptake. Cocaine binding sites in a fresh P₂ fraction of mouse striatum were labeled with [³H]CFT, a phenyltropane analog of cocaine also known as WIN 35,428, and compared with sites labeled with [³H]mazindol or [³H]GBR 12935. Under the conditions used, homogeneous binding was observed that was inhibited monophasically by cocaine, CFT, and mazindol; the same potencies were observed with the three radioligands. Saturation analysis in the presence and in the absence of unlabeled inhibitor (CFT, mazindol, cocaine) indicated a change in the K_d but not the B_max, consonant with a competitive mechanism. Tris-HCl reduced the affinity of each radioligand and unlabeled inhibitor without changing the B_max. N-Ethylmaleimide reduced the binding of all radioligands equally and cocaine offered protection. The dissociation rate of [³H]CFT and [³H]mazindol binding was not affected by the presence of mazindol and CFT, respectively. The B_max of [³H]CFT and [³H]mazindol binding was the same; the relatively higher value for [³H]GBR 12935 binding in analyses involving varying tritiated GBR 12935 only, was due primarily to an underestimation of the specific activity of [³H]GBR 12935. All results are in agreement with a one-site model in which cocaine, CFT, mazindol, and GBR 12935 share a common binding site in mouse striatum.     

3H]mazindol binding associated with neuronal dopamine and norepinephrine uptake sites

[3H]Mazindol labels neuronal dopamine uptake sites in corpus striatum membranes (KD = 18 nM) and neuronal norepinephrine uptake sites in cerebral cortex and submaxillary/sublingual gland membranes (KD = 4 nM). The potencies of various inhibitors of biogenic amine uptake in reducing [3H]mazindol binding in striatal membranes correlate with their potencies for inhibition of neuronal [3H]dopamine accumulation, whereas their potencies in reducing [3H]mazindol binding to cortical and salivary gland membranes correlate with their potencies for inhibition of neuronal [3H]norepinephrine accumulation. Similar to the dopamine and norepinephrine uptake systems, [3H]mazindol binding in all three tissues is dependent upon sodium (with potassium, lithium, rubidium, and Tris being ineffective substitutes) and chloride (with sulfate and phosphate being ineffective substitutes). In membranes of the cerebral cortex and salivary gland, half-maximal stimulation is observed at 50-80 mM NaCl, whereas in membranes of the corpus striatum half-maximal stimulation occurs at 240 mM NaCl. In striatal membranes NaCl increases the affinity of [3H]mazindol binding with no effect on the maximal number of sites. The enhancement of affinity is due to a selective slowing of the dissociation of the ligand from its binding site. The association of [3H]mazindol binding sites with neuronal dopamine uptake sites in the corpus striatum is further supported by the reduction of [3H]mazindol binding sites in striatal membranes following destruction of dopaminergic neurons by 6-hydroxydopamine. Similarly, the association of [3H]mazindol binding sites with neuronal norepinephrine uptake sites in cerebral cortex is supported by the reduction of [3H]mazindol binding to cortical membranes following destruction of noradrenergic neurons by N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine.     

Photoaffinity labeling of the dopamine reuptake carrier protein with 3-azido[3H]GBR-12935

A high affinity tritiated azido-diphenylpiperazine derivative, 3-azido[3H]GBR-12935, was synthesized as a potential photoaffinity probe of the dopamine transporter. Initially, the reversible binding of 3-azido[3H]GBR-12935 to crude synaptosomal membranes from the rat striatum was characterized. Specific binding was sodium dependent and inhibited by a variety of drugs that are known to potently inhibit dopamine uptake. Other neurotransmitter uptake inhibitors, as well as cis-flupenthixol, a potent inhibitor of [3H]GBR-12935 binding to piperazine binding sites, failed to inhibit specific binding at concentrations of less than or equal to 10 microM. A good correlation was observed between the relative potencies of these drugs in inhibiting dopamine uptake into synaptosomes and in inhibiting specific 3-azido[3H]GBR-12935 binding to rat striatal membranes (r = 0.95, p less than 0.01). These data suggest that 3-azido[3H]GBR-12935, like other diphenylpiperazines such as [3H]GBR-12935 and [3H]GBR-12909, binds primarily to the dopamine transporter under defined assay conditions. After UV photolysis of crude synaptosomal membranes preincubated with 3-azido[3H]GBR-12935 (1-2 nM), a single radiolabeled polypeptide with an apparent molecular mass of 80 kDa was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Photoincorporation of 3-azido[3H]GBR-12935 into this polypeptide was inhibited selectively by compounds that inhibit the uptake of dopamine (but not other biogenic amines) and was completely dependent on the presence of Na+. No photolabeled proteins were observed when cerebellar membranes were substituted for striatal membranes. Essentially complete adsorption of the radiolabeled 80-kDa polypeptide to wheat germ agglutinin and elution with N-acetyl-D-glucosamine strongly suggest that the dopamine transporter polypeptide photolabeled by 3-azido[3H]GBR-12935 is glycosylated.     

Binding characteristics of the dopamine uptake inhibitor [3H]nomifensine to striatal membranes

Binding of the radiolabeled antidepressant [3H]nomifensine to rat and rabbit striatal membranes has been characterized. The specific binding of [3H]nomifensine to striatal membranes was stable, reversible and saturable. Saturation experiments indicated that [3H]nomifensine labeled a single site with an affinity (Kd) of 80 nM and a total number of binding sites (Bmax) of 6.5 pmoles/mg protein both in rat and rabbit striatal membranes. The affinity constants obtained from kinetic analyses and competition experiments were in fairly good agreement with those obtained in saturation experiments. Compounds known to inhibit [3H]dopamine uptake in vitro, such as nomifensine, 4-hydroxy-nomifensine, mazindol, amfonelic acid and benztropine, were the most potent competitors of nomifensine binding. Additionally, the absolute potencies of various drugs in competing for [3H]nomifensine binding to rat and rabbit striatal membranes correlated closely with their potencies in inhibiting [3H]dopamine uptake into striatal synaptosomes. Specific [3H]nomifensine binding was dependent on the presence of NaCl which is also consistent with its association with the dopamine uptake pump. The number, but not the affinity, of striatal [3H]nomifensine binding sites was reduced significantly following in vivo lesions with 6-hydroxydopamine. The number of [3H]nomifensine binding sites was found to be highest in areas rich in dopamine nerve terminals such as the striatum and olfactory tubercle. These results suggest that [3H]nomifensine binds to a site on dopaminergic nerve terminals associated with the dopamine uptake pump.     

Synthesis, monoamine transporter binding, properties, and functional monoamine uptake activity of 3beta-[4-methylphenyl and 4-chlorophenyl]-2 beta-[5-(substituted phenyl)thiazol-2-yl]tropanes

Synthetic methods were developed for the synthesis of the 3beta-(4-substituted phenyl)-2 beta-[5-(substituted phenyl)thiazol-2-yl]tropanes (4a-s). The compounds were evaluated for their monoamine transporter binding and monoamine uptake inhibition properties using both rat brain tissue and cloned transporter assays. In general, the compounds showed higher dopamine transporter (DAT) affinity relative to the serotonin and norepinephrine transporters (SERT and NET, respectively) and greater [3H]dopamine uptake inhibition potency relative to [3H]serotonin and [3H]norepinephrine uptake inhibition. Several compounds were DAT selective relative to the SERT and NET in the monoamine transporter binding assays. The most potent and selective analog in the functional monoamine uptake inhibition test was 3beta-(4-methylphenyl-2 beta-[5-(3-nitrophenyl)thiazol-2-yl]tropane (4p).     

Oxygenated analogues of 1-[2-(Diphenylmethoxy)ethyl]- and 1-[2-[Bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazines (GBR 12935 and GBR 12909) as potential extended-action cocaine-abuse therapeutic agents

An investigation into the preparation of potential extended-release cocaine-abuse therapeutic agents afforded a series of compounds related to 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (1a) and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (1b) (GBR 12935 and GBR 12909, respectively), which were designed, synthesized, and evaluated for their ability to bind to the dopamine transporter (DAT) and to inhibit the uptake of [(3)H]-labeled dopamine (DA). The addition of hydroxy and methoxy substituents to the benzene ring on the phenylpropyl moiety of 1a-1d resulted in a series of potent and selective ligands for the DAT (analogues 5-28). The hydroxyl groups were included to incorporate a medium-chain carboxylic acid ester into the molecules, to form oil-soluble prodrugs, amenable to "depot" injection techniques. The introduction of an oxygen-containing functionality to the propyl side chain provided ketones 29 and 30, which demonstrated greatly reduced affinity for the DAT and decreased potency in inhibiting the uptake of [(3)H]DA, and benzylic alcohols 31-36, which were highly potent and selective at binding to the DAT and inhibiting [(3)H]DA uptake. The enantiomers of 32 (34 and 36) were practically identical in biological testing. Compounds 1b, 32, 34, and 36 all demonstrated the ability to decrease cocaine-maintained responding in monkeys without affecting behaviors maintained by food, with 34 and 36 equipotent to each other and both more potent in behavioral tests than the parent compound 1b. Intramuscular injections of compound 41 (the decanoate ester of racemate 32) eliminated cocaine-maintained behavior for about a month following one single injection, without affecting food-maintained behavior. The identification of analogues 32, 34, and 36, thus, provides three potential candidates for esterification and formulation as extended-release cocaine-abuse therapeutic agents.     

Metaphit prevents locomotor activation induced by various psychostimulants and interferes with the dopaminergic system in mice

Metaphit, an isothiocyanate analog of phencyclidine and a proposed phencyclidine receptor acylator, inactivated the carrier involved in the neuronal uptake of dopamine in in vitro experiments with preparations of the striatum in the mouse. In ex vivo experiments 2 and 24 hr after the intravenous administration of metaphit, no changes were observed either in the binding of [3H]cocaine to striatal membranes or in the uptake of [3H]dopamine into synaptosomes or slices. In in vivo experiments 24 hr after pretreatment with metaphit, selective labelling of uptake sites for dopamine in the striatum of the mouse with [3H]GBR 12935 was unaffected. In these in vivo experiments, however, metaphit antagonized the locomotor stimulation induced by blockers of the uptake of dopamine (methylphenidate, mazindol, cocaine, GBR 12909) but not that induced by drugs that affect locomotion by other mechanisms (amphetamine, phencyclidine). Twenty-four hours after treatment with metaphit there was an increase in homovanillic acid in all regions of the brain studied (striatum, olfactory tubercle, cerebral cortex). There was no effect of metaphit on the disappearance rate of 3,4-dihydroxyphenylacetic acid and homovanillic acid from the striatum during the inhibition of monoamine oxidase with pargyline. If the increase in homovanillic acid reflected a greater rate of dopamine catabolism in metaphit-treated mice, it could explain the lack of locomotor stimulation of blockers uptake of the dopamine in these animals, resulting from a rapid breakdown of extracellularly accumulated dopamine.     

Novel 3-aminomethyl- and 4-aminopiperidine analogues of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazines: synthesis and evaluation as dopamine transporter ligands

We have undertaken a program to develop cocaine antagonists based on the premise that such compounds should block cocaine binding but permit reuptake of dopamine at the dopamine transporter (DAT). To evaluate the structural features of potential cocaine antagonists, 3-aminomethylpiperidine and 4-aminopiperidine moieties were incorporated at the central bridge region (piperazine ring) of GBR 12935. The compounds were assayed as inhibitors of [(125)I]RTI-55 binding at the DAT and monoamine transport. The results indicated that most of the new compounds preferentially inhibited norepinephrine reuptake by its transporter (NET) but in some cases retained binding selectivity for the DAT. In general, the binding selectivity and potency of [(3)H]NE reuptake inhibition were very sensitive to modifications of the central bridge diamine moiety (position of two basic nitrogen atoms). Compound 6 exhibited the highest ratio (14-fold) of DA reuptake inhibition to RTI-55 binding inhibition at the DAT; however, in an in vitro assay of cocaine antagonism, this compound failed to reduce inhibition of [(3)H]DA uptake by cocaine. These results demonstrated that separation of biological activities into the binding and reuptake inhibition can be achieved by alterations in the internal diamine component of GBR 12935, but additional modifications are necessary before these agents constitute lead compounds for development as cocaine antagonists.     

3H]cocaine binding and inhibition of [3H]dopamine uptake is similar in both the rat striatum and nucleus accumbens

Cocaine binds with high affinity to the dopamine transporter in both the striatum and the nucleus accumbens. We examined Na(+)-dependent [3H]cocaine binding, mazindol inhibition of [3H]cocaine binding and cocaine inhibition of [3H]dopamine uptake in both rat brain areas. The striatum and nucleus accumbens demonstrated Na(+)-dependent [3H]cocaine binding with similar densities. Mazindol inhibited [3H]cocaine binding with a similar IC50 in both the striatum and nucleus accumbens. Likewise, cocaine inhibited [3H]dopamine uptake in both brain regions with equivalent efficacy. From these data we conclude that the dopamine transporter is similar in both the striatum and nucleus accumbens.     

Development of long-acting dopamine transporter ligands as potential cocaine-abuse therapeutic agents: chiral hydroxyl-containing derivatives of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine and 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine

In our search for long-acting agents for the treatment of cocaine abuse, a series of optically pure hydroxylated derivatives of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (1) and 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (2) (GBR 12909 and GBR 12935, respectively) were synthesized and evaluated in vitro and in vivo. The enantiomers of the 2-hydroxylated analogues displayed substantial enantioselectivity. The S enantiomers displayed higher dopamine transporter (DAT) affinity and the R enantiomers were found to interact at the serotonin transporter (SERT) with higher affinity. The two-carbon spacer between the hydroxyl group and the piperazine ring was essential for enantioselectivity, and the length of the alkyl chain between the phenyl group and the piperazine ring influenced binding affinity and selectivity for the DAT and SERT. Phenylethyl analogues had a higher binding affinity for the SERT and a weaker affinity and selectivity for the DAT than the corresponding phenylpropyl analogues. Thus, (S)-(+)-1-[4-[2-[bis(4-fluorophenyl)methoxy]ethyl]piperazinyl]-3-phenylpropan-2-ol (6) displayed the highest affinity to the DAT, and (S)-(+)-1-[4-[2-(diphenylmethoxy)ethyl]piperazinyl]-3-phenylpropan-2-ol (8) had the highest selectivity. The latter (8) is one of the most DAT selective ligands known. In accord with the in vitro data, 6 showed greater potency than 7 in elevating extracellular dopamine levels in a microdialysis assay and in inhibiting cocaine-maintained responding in rhesus monkeys.     

Effects of phencyclidine on [3H]catecholamine and [3H]serotonin uptake in synaptosomal preparations from rat brain

Phencyclidine inhibited uptake in vitro of [³H]norepinephrine (ic₅₀ 0.52 μM), [³H]dopamine (ic₅₀ 0.73 μM) and [³H]serotonin (ic₅₀ 0.80 μM) in crude synaptosomal preparations from rat brain through a competitive mechanism. Phencyclidine was fairly similar in potency to d-amphetamine and methylphenidate in inhibiting catecholamine uptake but was 8 times more potent than d-amphetamine and 34 times more potent than methylphenidate in inhibiting [³H]serotonin uptake.     

Modulation by dopamine of [3H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine ([3H]BTCP, a phencyclidine derivative) binding to the dopamine uptake complex.

The modulation by dopamine of the binding of [3H]BTCP to the dopamine (DA) uptake complex was investigated in vivo (in control, reserpine- and L-DOPA-treated mice) and in vitro (on membrane preparations of the striatum of the rat). In both cases increasing doses of DA exerted a non-competitive inhibition of binding of [3H]BTCP, with a Ki value close to its K0.5, determined in competition experiments. Amphetamine and cocaine were also non-competitive inhibitors of the binding of [3H]BTCP, while GBR 12783 was competitive. In the presence of DA, the amount of cocaine required to inhibit the binding of [3H]BTCP was increased both in vitro and in vivo. These results suggest that inhibitors of the uptake of DA, such as BTCP or GBR 12783, modulate allosterically the uptake of DA, by binding to a site different from the DA recognition site. Cocaine, however, seems to share the same recognition site as DA.     

Synthesis and transporter binding properties of bridged piperazine analogues of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR 12909)

A series of analogues related to 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (2) and 1-?2-[bis(4-fluorophenyl)methoxy]ethyl?-4-(3-phenylpropyl)piperazine (3) (GBR 12935 and GBR 12909, respectively), in which the piperazine moiety was replaced by bridged piperazines for structural rigidity, has been designed, synthesized, and evaluated for their ability to bind to the dopamine transporter (DAT) and to inhibit the uptake of (3)H-labeled dopamine (DA). The binding data indicated that compounds 7 and 11, the N-methyl- and N-propylphenyl-3,8-diaza[3.2. 1]bicyclooctane analogues of 3, showed high affinity for the DAT (IC(50) = 8.0 and 8.2 nM, respectively), and 7 had high selectivity at the DAT relative to the serotonin transporter (SERT) (88- and 93-fold for binding and reuptake, respectively). They also displayed linear activity in DA uptake inhibition, possessing a similar binding and reuptake inhibition profile to 3. The N-indolylmethyl analogue 16 showed the highest affinity (IC(50) = 1.4 nM) of the series, with a 6-fold increase over its corresponding N-phenypropyl derivative 11. Interestingly, this compound exhibited a high ratio (29-fold) of IC(50) for the inhibition of DA reuptake versus binding to the DAT. Replacing the piperazine moiety of 2 and 3 with (1S, 4S)-2,5-diazabicyclo[2.2.1]heptane resulted in compounds 23-26, which showed moderate to poor affinity (IC(50) = 127-1170 nM) for the DAT. Substitution of the homopiperazine moiety of 4 with a more rigid 3,9-diazabicyclo[4.2.1]nonane gave compounds 28-33. However, the binding data showed that compound 32 displayed a 10-fold decrease in affinity at the DAT and a 100-fold decrease in selectivity at the DAT relative to the SERT compared to its corresponding homopiperazine compound 4.     

Transporter-mediated actions of R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane

R-(-)-1-(Benzofuran-2-yl)-2-propylaminopentane [(-)-BPAP] is a catecholaminergic and serotonergic activity enhancer that increases impulse-evoked catecholamine and serotonin release from nerve terminals, and is a candidate for symptomatic treatment of early Parkinson's disease. We now report the catecholamine and serotonin transporter-mediated actions of (-)-BPAP. The effects of (-)-BPAP on inhibition of neurotransmitter uptake and radioligand binding were assessed using human embryonic kidney 293 cells (HEK 293 cells) expressing cDNA for the human dopamine transporter (hDAT), norepinephrine transporter (hNET), and serotonin transporter (hSERT). The IC(50) values for the effects of (-)-BPAP on [3H]dopamine, [3H]norepinephrine, and [3H]serotonin uptake were 42+/-9, 52+/-19, and 640+/-120 nM, respectively. The IC(50) values for the effects of (-)-BPAP on [125I]3 beta-(4-iodophenyl)tropane-2 beta-carboxylic acid methyl ester ([125I]RTI-55) binding to hDAT, hNET, and hSERT were 16+/-2, 211+/-61, and 638+/-63 nM, respectively. The effects of (-)-BPAP on spontaneous and tyramine-induced norepinephrine and dopamine release from rat brain synaptosomes using a superfusion system were also assessed. Tyramine but not (-)-BPAP potentiated norepinephrine release. Furthermore, (-)-BPAP inhibited tyramine-induced norepinephrine release. Thus, (-)-BPAP may block tyramine-induced adverse effects such as hypertensive crisis. The actions of (-)-BPAP on the spontaneous and tyramine-induced dopamine release resembled its effects on norepinephrine release. We conclude that (-)-BPAP is not only catecholaminergic and serotonergic activity enhancer, but also a norepinephrine and dopamine uptake inhibitor and a weak serotonin uptake inhibitor that does not possess a tyramine-like action on catecholamine release, and is an inhibitor of tyramine-induced release of norepinephrine.     

In vivo labelling of the neuronal dopamine uptake complex in the mouse striatum by [3H]GBR 12783.

Various characteristics of the in vivo striatal binding of [3H]GBR 12783 (1-[2-(diphenylmethoxy)-ethyl]-4-(3-phenyl-1[3H]-2-propenyl)pipera zine), a specific ligand of the neuronal dopamine uptake complex, were determined in mice. Increasing doses of the ligand revealed the saturability of the binding at a single site with half-maximal saturation at a dose of approximately 7 mumol/kg and an apparent maximal number of binding sites (Bmax) of 12.8 pmol/mg protein in striatum. Specific binding was prevented by various dopamine uptake blockers, pyrovalerone, GBR 13069, GBR 12783, N-[1-2-benzo(b)thiophenyl)cyclohexyl] piperidine, cocaine, methylphenidate and was inhibited in a stereoselective manner by the enantiomers of nomifensine. Other drugs which are not dopamine uptake blockers either did not modify [3H]GBR 12783 binding (the diphenylbutylpiperazine derivative flupenthixol) or increased it (the diphenylpiperazine derivative flunarizine or the chemically unrelated compounds fenfluramine and SKF 525A). A close correlation was found between occupancy of the striatal [3H]GBR 12783 binding site and the stimulant locomotor effect of the drug. A similar specific striatal binding of [3H]GBR 12783 was evidenced in both NMRI and CD1 strains. It was concluded that [3H]GBR 12783 administered in vivo provides a measure of the density of dopamine uptake sites in mouse striatum.