N-substituted benztropine analogs: selective dopamine transporter ligands with a fast onset of action and minimal cocaine-like behavioral effects

Previous studies suggested that differences between the behavioral effects of cocaine and analogs of benztropine were related to the relatively slow onset of action of the latter compounds. Several N-substituted benztropine analogs with a relatively fast onset of effects were studied to assess whether a fast onset of effects would render the effects more similar to those of cocaine. Only one of the compounds increased locomotor activity, and the increases were modest compared with those of 10 to 20 mg/kg cocaine. In rats trained to discriminate 10 mg/kg cocaine from saline none of the compounds produced more than 40% cocaine-like responds up to 2 h after injection. None of the compounds produced place-conditioning when examined up to 90 min after injection, indicating minimal abuse liability. The compounds had 5.6 to 30 nM affinities at the dopamine transporter (DAT), with uniformly lower affinities at norepinephrine and serotonin transporters (from 490-4600 and 1420-7350 nM, respectively). Affinities at muscarinic M(1) receptors were from 100- to 300-fold lower than DAT affinities, suggesting minimal contribution of those sites to the behavioral effects of the compounds. Affinities at histaminic H(1) sites were from 11- to 43-fold lower than those for the DAT. The compounds also had affinity for sigma, 5-hydroxytryptamine(1) (5-HT(1)), and 5-HT(2) receptors that may have contributed to their behavioral effects. Together, the results indicate that a slow onset of action is not a necessary condition for reduced cocaine-like effects of atypical DAT ligands and suggest several mechanisms that may contribute to the reduced cocaine-like efficacy of these compounds.     

Effects of N-substituted analogs of benztropine: diminished cocaine-like effects in dopamine transporter ligands

Previous studies demonstrated that analogs of benztropine (BZT) possess high affinity for the dopamine transporter, inhibit dopamine uptake, but generally have behavioral effects different from those of cocaine. One hypothesis is that muscarinic-M(1) receptor actions interfere with cocaine-like effects. Several tropane-nitrogen substitutions of 4',4"-diF-BZT have reduced M(1) affinity compared with the CH(3)-analog (AHN 1-055; 3alpha-[bis-(4-fluorophenyl)methoxy]tropane). All of the compounds displaced [(3)H]WIN 35,428 (2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane) binding with affinities ranging from 11 to 108 nM. Affinities at norepinephrine ([(3)H]nisoxetine) and serotonin ([(3)H]citalopram) transporters ranged from 457 to 4810 and 376 to 3260 nM, respectively, and at muscarinic M(1) receptors ([(3)H]pirenzepine) from 11.6 (AHN 1-055) to higher values, reaching 1030 nM for the other BZT-analogs. Cocaine and AHN 1-055 produced dose-related increases in locomotor activity in mice, with AHN 1-055 less effective than cocaine. The other compounds were ineffective in stimulating activity. In rats discriminating cocaine (29 micromol/kg i.p.) from saline, WIN 35,428 fully substituted for cocaine, whereas AHN 1-055 produced a maximal substitution of 79%. None of the other analogs fully substituted for cocaine. WIN 35,428 produced dose-related leftward shifts in the cocaine dose-effect curve, whereas selected BZT analogs produced minimal changes in the effects of cocaine. The results suggest that reducing M(1) affinity of 4',4"-diF-BZT with N-substitutions reduces effectiveness in potentiating the effects of cocaine. Furthermore, although the BZT-analogs bind with high affinity at the dopamine transporter, their behavioral effects differ from those of cocaine. These compounds have reduced efficacy compared with cocaine, a long duration of action, and may serve as leads for the development of medications to treat cocaine abuse.     

Place conditioning and locomotor effects of N-substituted, 4',4''-difluorobenztropine analogs in rats

Previous studies demonstrated that analogs of benztropine [3alpha-(diphenyl-methoxy)tropane (BZT)] bind to the dopamine (DA) transporter with high affinity, inhibit DA uptake, but do not maintain rates of responding in self-administration procedures comparable with those maintained by cocaine. Some BZT analogs have an onset of action that is slower than that for cocaine that may contribute to this decreased effectiveness. In addition, some BZT analogs have affinity for muscarinic-M1 receptors that may interfere with reinforcing effects. The present study assessed effects of BZT analogs in place-conditioning procedures designed to accommodate variations in onset of effect. BZT analogs with variations in relative affinities for the DA transporter over M1 receptors from equal [AHN 1-055 (3alpha-[bis(4'-fluorophenyl)methoxy]-tropane)] to 16-fold [JHW 007 (N-(n-butyl)-3alpha-[bis(4'-fluorophenyl)methoxy]-tropane)] were compared with cocaine and the muscarinic antagonist, atropine. Cocaine (10-20 mg/kg) but not atropine (1.0-5.6 mg/kg) produced dose-related place conditioning. The N-methyl-substituted BZT analog, AHN 1-055, was without significant effects at doses that ranged from 0.3 to 3.0 mg/kg and when administered up to 90 min before conditioning trials. In contrast, effects of AHN 2-005 (N-allyl-3alpha-[bis(4'-fluorophenyl)methoxy]-tropane; 0.1-10.0 mg/kg) were significant, and those of JHW 007 approached significance when administered 45 min but not immediately or 90 min before trials. Atropine blocked the effect of AHN 2-005 and approached significant antagonism of cocaine. The present study further supports and extends previous results showing minimal preclinical indications of abuse liability of BZT analogs and suggests that these differences from cocaine are not entirely accounted for by a slower onset of action or muscarinic M1 receptor affinity.     

Applicability of the dopamine and rate hypotheses in explaining the differences in behavioral pharmacology of the chloro-benztropine analogs: studies conducted using intracerebral microdialysis and population pharmacodynamic modeling

Previous studies indicated that the chloro-benztropine analogs differed significantly in their cocaine-like activity, which was not expected based on the similarity in their in vitro binding affinity and functional potency at the dopamine transporter (DAT). The present study was designed to extend the understanding of the involvement of both pharmacokinetic and pharmacodynamic factors in mediating the behavioral differences among these analogs. The pharmacokinetics of 3'-chloro-3alpha-(diphenylmethoxy)tropane (3'-Cl BZT), the analog showing a cocaine-like behavioral profile in rodents, was compared with previously reported pharmacokinetic characteristics of cocaine and 4',4'-dichloro-3alpha-(diphenylmethoxy)tropane (4',4'-diCl BZT), an analog totally devoid of cocaine-like actions. Microdialysis studies in rats were conducted to determine whether 3'-Cl and 4',4'-diCl BZT differed significantly in their effect on nucleus accumbens extracellular dopamine levels, with cocaine serving as a reference. A mechanistic model based on DAT association/dissociation kinetics was used to describe the time delay between the plasma concentrations of the chloro-analogs and their dopaminergic effects. 3'-Cl BZT had plasma elimination half-life of 1.9 h versus 0.5 and 21.1 h for cocaine and 4',4'-diCl BZT, respectively. 4',4'-diCl BZT increased the DA levels at a slower rate and to a significantly lower extent relative to 3'-Cl BZT that were, in turn, lower than cocaine. The duration of dopamine elevation was as follows: 4',4'-diCl BZT > 3'-Cl BZT > cocaine. The model indicated faster association and dissociation with DAT for 3'-Cl BZT relative to 4',4'-diCl BZT. The present results indicate that behavioral differences among the chloro-analogs may be explainable based on both the dopamine and rate hypotheses of drug abuse.     

Relationship between in vivo occupancy at the dopamine transporter and behavioral effects of cocaine, GBR 12909 [1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine], and benztropine analogs

Analogs of benztropine (BZT) bind to the dopamine (DA) transporter and inhibit DA uptake but often have behavioral effects that differ from those of cocaine and other DA-uptake inhibitors. To better understand these differences, we examined the relationship between locomotor-stimulant effects of cocaine, 1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)-piperazine (GBR 12909), and BZT analogs [(3alpha-[bis(4'-fluorophenyl)methoxy]-tropane) (AHN 1-055) and (N-allyl-3alpha-[bis(4'-fluorophenyl)methoxy]-tropane) (AHN 2-005)] and their in vivo displacement of the DA transporter ligand [125I]3beta-(4-iodophenyl)-tropan-2beta-carboxylic acid isopropyl ester hydrochloride (RTI-121) in striatum. Cocaine, GBR 12909, and BZT analogs each displaced [125I]RTI-121 and stimulated locomotor activity in a dose- and time-dependent manner. The time course revealed a slower onset of both effects for AHN 1-055 and AHN 2-005 compared with cocaine and GBR 12909. The BZT analogs were less effective than cocaine and GBR 12909 in stimulating locomotor activity. Locomotor stimulant effects of cocaine were generally greater than predicted by the regression of displacement of [125I]RTI-121 and effect at short times after injection and less than predicted at longer times after injection. This result suggests that the apparent rate of occupancy of the DA transporter, in addition to percentage of sites occupied, contributes to the behavioral effects of cocaine. The present results suggest that among drugs that act at the DA transporter, the slower apparent rates of occupancy with the DA transporter by the BZT analogs may contribute in an important way to differences in their effectiveness.     

Biochemical and behavioral characterization of novel methylphenidate analogs

As part of a project to develop treatment agents for cocaine abuse, (+/-)-threo-methylphenidate (TMP) and 11 analogs were characterized biochemically and behaviorally to assess their potential as anti-cocaine medications. The compounds contained aryl and/or nitrogen substitutions, and/or replacement of the ester function by an alcohol or ether. All of the analogs, except for the N-methyl-substituted compounds, showed increased inhibitory potency against (3)H-(-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate ([(3)H]WIN 35,428) ([(3)H]WIN) binding to the dopamine transporter, compared with TMP. In general, parallel results were obtained for inhibition of [(3)H]dopamine ([(3)H]DA) uptake. Although compounds with N-substitutions were proportionally less potent at blocking DA uptake than WIN binding (compared with the unsubstituted compounds), one such compound that was 6-fold more potent against [(3)H]WIN binding than [(3)H]DA uptake did not attenuate inhibition by cocaine of synaptosomal [(3)H]DA transport. The compounds were significantly less potent in displacing [(3)H]citalopram binding from the serotonin transporter. In cocaine discrimination studies in rats, all but two of the analogs (both N-substituted) completely generalized with the cocaine stimulus. Robust positive correlations were observed between potency in the drug discrimination assay and activity at the dopamine transporter, but not the serotonin transporter. When tested for their ability to alter cocaine discrimination, four of the analogs (three of which had N-substitutions and shallow dose-response curves as cocaine substitutes) actually enhanced cocaine discrimination, often at combined doses of cocaine and test compound that were inactive when given separately. Taken together, the results suggest that TMP analogs may have potential as substitution therapies for the treatment of cocaine abuse.     

Effects of 4'-chloro-3 alpha-(diphenylmethoxy)-tropane on mesostriatal, mesocortical, and mesolimbic dopamine transmission: comparison with effects of cocaine

Increase in dopamine (DA) neurotransmission resulting from blockade of the DA transporter (DAT) after administration of cocaine is believed to play a major role in mediating its behavioral and reinforcing effects. Since it was hypothesized that drugs that block the DAT have cocaine-like behavioral effects, it was of interest to study in the present article the stimulant effects of cocaine on locomotor activity and on pattern of activation of DA neurotransmission in different DAergic terminal areas in rats and compare these effects with those of 4'-chloro-3alpha-(diphenylmethoxy)-tropane (4-Cl-BZT), a benztropine analog showing higher affinity for the DAT, but reduced behavioral effects compared with cocaine. Administration of cocaine resulted in a dose-dependent stimulation of locomotor activity and DA neurotransmission in the nucleus accumbens shell and core, dorsal caudate, and in the medial prefrontal cortex (PFCX) measured by microdialysis. At comparable doses, the effects of 4-Cl-BZT on DA levels in all brain areas except the PFCX were generally reduced compared with those of cocaine, as were the effects on locomotor activity. The differences in behavioral effects corresponded generally to differences between the drugs with regard to their stimulation of extracellular DA levels, although the mechanism(s) for the differences in extracellular DA may involve effects mediated by sites other than the DAT or differences in the efficiency of the two drugs in blocking DA uptake. Nonetheless, the present results suggest that the differences in behavioral effects between cocaine and 4-Cl-BZT are related to differences in their patterns of activation of DA transmission.     

Cocaine-induced increases in vesicular dopamine uptake: role of dopamine receptors

The vesicular monoamine transporter-2 is the sole transporter responsible for sequestration of monoamines, including dopamine (DA), into synaptic vesicles. Previous studies demonstrate that agents that inhibit DA transporter function, such as cocaine, increase vesicular [(3)H]DA uptake and binding of the ligand [(3)H]dihydrotetrabenazine ([(3)H]DHTBZ), as assessed in vesicles prepared from treated rats. The present studies examine the role of DA receptors in these cocaine-induced effects. Results demonstrate that administration of the D(2) DA receptor antagonist, eticlopride, but not the D(1) DA receptor antagonist, SCH23390, inhibited these cocaine-induced increases. Similar to the effects of cocaine, treatment with the D(2) agonist, quinpirole, increased both vesicular [(3)H]DA uptake and [(3)H]DHTBZ binding. In contrast, administration of the D(1) agonist, SKF81297, was without effect on vesicular [(3)H]DA uptake or [(3)H]DHTBZ binding. Finally, coadministration of quinpirole and cocaine did not further increase vesicular [(3)H]DA uptake or [(3)H]DHTBZ binding when compared with treatment with either agent alone. These data suggest that cocaine-induced increases in vesicular DA uptake and DHTBZ binding are mediated by a D(2) receptor-mediated pathway. Furthermore, results indicate that D(2) receptor activation, per se, is sufficient to increase vesicular DA uptake.     

meso-Transdiene analogs inhibit vesicular monoamine transporter-2 function and methamphetamine-evoked dopamine release

Lobeline, a nicotinic receptor antagonist and neurotransmitter transporter inhibitor, is a candidate pharmacotherapy for methamphetamine abuse. meso-Transdiene (MTD), a lobeline analog, lacks nicotinic receptor affinity, retains affinity for vesicular monoamine transporter 2 (VMAT2), and, surprisingly, has enhanced affinity for dopamine (DA) and serotonin transporters [DA transporter (DAT) and serotonin transporter (SERT), respectively]. In the current study, MTD was evaluated for its ability to decrease methamphetamine self-administration in rats relative to food-maintained responding. MTD specifically decreased methamphetamine self-administration, extending our previous work. Classical structure-activity relationships revealed that more conformationally restricted MTD analogs enhanced VMAT2 selectivity and drug likeness, whereas affinity at the dihydrotetrabenazine binding and DA uptake sites on VMAT2 was not altered. Generally, MTD analogs exhibited 50- to 1000-fold lower affinity for DAT and were equipotent or had 10-fold higher affinity for SERT, compared with MTD. Representative analogs from the series potently and competitively inhibited [(3)H]DA uptake at VMAT2. (3Z,5Z)-3,5-bis(2,4-dichlorobenzylidene)-1-methylpiperidine (UKMH-106), the 3Z,5Z-2,4-dichlorophenyl MTD analog, had improved selectivity for VMAT2 over DAT and importantly inhibited methamphetamine-evoked DA release from striatal slices. In contrast, (3Z,5E)-3,5-bis(2,4-dichlorobenzylidene)-1-methylpiperidine (UKMH-105), the 3Z,5E-geometrical isomer, inhibited DA uptake at VMAT2, but did not inhibit methamphetamine-evoked DA release. Taken together, these results suggest that these geometrical isomers interact at alternate sites on VMAT2, which are associated with distinct pharmacophores. Thus, structural modification of the MTD molecule resulted in analogs exhibiting improved drug likeness and improved selectivity for VMAT2, as well as the ability to decrease methamphetamine-evoked DA release, supporting the further evaluation of these analogs as treatments for methamphetamine abuse.     

Regulation of the vesicular monoamine transporter-2: a novel mechanism for cocaine and other psychostimulants

The plasmalemmal dopamine (DA) transporter (DAT) is a principal site of action for cocaine. This report presents the novel finding that in addition to inhibiting DAT function, cocaine administration rapidly alters vesicular DA transport. Specifically, cocaine treatment abruptly and reversibly increased both the V(max) of DA uptake and the B(max) of vesicular monoamine transporter-2 (VMAT-2) ligand (dihydrotetrabenazine) binding, as assessed ex vivo in purified rat striatal synaptic vesicles. Selective inhibitors of the DAT (amfonelic acid and GBR12935), but not the plasmalemmal serotonin transporter (fluoxetine), also increased vesicular DA uptake. Moreover, DA depletion resulting from administration of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine had cocaine-like effects. Conversely, administration of the DA-releasing agent methamphetamine rapidly decreased vesicular uptake. Taken together, these data demonstrate for the first time ex vivo that cocaine treatment rapidly alters vesicular monoamine transport, and suggest that alterations in cytoplasmic DA concentrations contribute to stimulant-induced changes in vesicular DA uptake. Hence, the VMAT-2 may be an important target for developing strategies to treat not only cocaine addiction but also other disorders involving alterations in neuronal DA disposition, including Parkinson's disease.     

The phencyclidine (PCP) analog N-[1-(2-benzo(B)thiophenyl) cyclohexyl]piperidine shares cocaine-like but not other characteristic behavioral effects with PCP, ketamine and MK-801

Phencyclidine (PCP) inhibits dopamine (DA) uptake and acts as a noncompetitive N-methyl-D-aspartate antagonist by binding to PCP receptors. The PCP analog N-[1-(2-benzo(b)thiophenyl) cyclohexyl]piperidine (BTCP, GK13) is a potent DA uptake inhibitor, but has low affinity for PCP receptors. The behavioral effects of BTCP were compared with those of PCP, ketamine, MK-801 and cocaine. In mice, BTCP, like cocaine, produced locomotion, sniffing and gnawing; haloperidol blocked these effects. PCP, ketamine and MK-801 produced locomotion, sniffing, swaying and falling. PCP, ketamine and MK-801 produced generalization in rats discriminating either cocaine, PCP or MK-801 from saline. Like cocaine, BTCP produced generalization in cocaine-discriminating rats only; haloperidol partially antagonized this effect. In pigeons, PCP-like catalepsy was produced by ketamine and MK-801, but not by BTCP. N-methyl-D-aspartate-induced convulsions in mice were antagonized by PCP, ketamine and MK-801, but not by BTCP or cocaine. Thus, BTCP shared only cocaine-like behavioral effects with PCP, ketamine and MK-801. A DA antagonist reduced the effects of BTCP. Therefore, the cocaine-like behavioral effects of BTCP may be mediated primarily by DA uptake mechanisms. However, PCP receptors, but not DA uptake mechanisms, may mediate the cocaine-like behavioral effects of PCP, ketamine and MK-801, because their order of potency in producing these effects (MK-801 greater than PCP greater than ketamine) is consistent with their potency order at PCP receptors, but not at DA uptake sites.     

Studies of the biogenic amine transporters. 14. Identification of low-efficacy "partial" substrates for the biogenic amine transporters

Several compounds have been identified that display low-efficacy, "partial substrate" activity. Here, we tested the hypothesis that the mechanism of this effect is a slower rate of induced neurotransmitter efflux than that produced by full substrates. Biogenic amine transporter release assays were carried out in rat brain synaptosomes and followed published procedures. [(3)H]1-methyl-4-phenylpyridinium (MPP(+)) was used to assess release from dopamine (DA) and norepinephrine nerve terminals, whereas [(3)H]5-hydroxytryptamine (5-HT) was used to assess release from 5-HT nerve terminals. A detailed time-course evaluation of DA transporter (DAT)-mediated efflux was conducted by measuring the efflux of [(3)H]MPP(+) after the addition of various test compounds. In vivo microdialysis experiments compared the effects of the full substrates [(±)-1-(2-naphthyl)propan-2-amine (PAL-287) and (S)-N-methyl-1-(2-naphthyl)propan-2-amine (PAL-1046)], to that of a partial DAT/5-HT transporter substrate [(S)-N-ethyl-1-(2-naphthyl)propan-2-amine (PAL-1045)] on extracellular DA and 5-HT in the nucleus accumbens of the rat. The in vitro release assays demonstrated that partial substrate activity occurs at all three transporters. In the DAT efflux experiments, D-amphetamine (full substrate) promoted a fast efflux (K1 = 0.24 min(-1)) and a slow efflux (K2 = 0.008 min(-1)). For the partial DAT substrates, K1 = ～0.04 min(-1), and K2 approximated zero. The in vivo microdialysis experiments showed that the partial substrate (PAL-1045) was much less effective in elevating extracellular DA and 5-HT than the comparator full substrates. We conclude that low-efficacy partial DAT substrates promote efflux at a slower rate than full substrates, and "partiality" reflects the ultra-slow K2 constant, which functionally limits the ability of these compounds to increase extracellular DA. We speculate that partial biogenic amine transporter substrates bind to the transporter but are less effective in inducing conformational changes required for reverse transport activity.     

Decreases in cocaine self-administration with dual inhibition of the dopamine transporter and σ receptors

Sigma receptor (σR) antagonists attenuate many behavioral effects of cocaine but typically not its reinforcing effects in self-administration procedures. However, the σR antagonist rimcazole and its N-propylphenyl analogs, [3-(cis-3,5-dimethyl-4-[3-phenylpropyl]-1-piperazinyl)-propyl]diphenylamine hydrochloride (SH 3-24) and 9-[3-(cis-3,5-dimethyl-4-[3-phenylpropyl]-1-piperazinyl)-propyl]carbazole hydrobromide (SH 3-28), dose-dependently decreased the maximal rates of cocaine self-administration without affecting comparable responding maintained by food reinforcement. In contrast, a variety of σR antagonists [N-phenethylpiperidine oxalate (AC927), N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine dihydrobromide (BD 1008), N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine dihydrobromide (BD 1047), N-[2-(3,4-dichlorophenyl) ethyl]-4-methylpiperazine dihydrochloride (BD 1063), and N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100)] had no effect on cocaine self-administration across the range of doses that decreased rates of food-maintained responding. Rimcazole analogs differed from selective σR antagonists in their dual affinities for σRs and the dopamine transporter (DAT) assessed with radioligand binding. Selective DAT inhibitors and σR antagonists were studied alone and in combination on cocaine self-administration to determine whether actions at both σRs and the DAT were sufficient to reproduce the effects of rimcazole analogs. Typical DAT inhibitors [2β-carbomethoxy-3β-(4-fluorophenyl)tropane (WIN 35,428), methylphenidate, and nomifensine] dose-dependently shifted the cocaine dose-effect curve leftward. Combinations of DAT inhibitor and σR antagonist doses that were behaviorally inactive alone decreased cocaine self-administration without effects on food-maintained responding. In addition, whereas the DAT inhibitors were self-administered at rates similar to those of cocaine, neither rimcazole analogs nor typical σR antagonists (NE-100 and AC927) maintained responding above control levels across a wide range of doses. These findings suggest that the unique effects of rimcazole analogs are due to dual actions at the DAT and σRs and that a combined target approach may have utility in development of medical treatments for cocaine abuse.     

Bupropion inhibits nicotine-evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine and from rat hippocampal slices preloaded with [(3)H]norepinephrine

Bupropion, an efficacious antidepressant and smoking cessation agent, inhibits dopamine and norepinephrine transporters (DAT and NET, respectively). Recently, bupropion has been reported to noncompetitively inhibit alpha3beta2, alpha3beta4, and alpha4beta2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes or established cell lines. The present study evaluated bupropion-induced inhibition of native alpha3beta2* and alpha3beta4* nAChRs using functional neurotransmitter release assays, nicotine-evoked [(3)H]overflow from superfused rat striatal slices preloaded with [(3)H]dopamine ([(3)H]DA), and nicotine-evoked [(3)H]overflow from hippocampal slices preloaded with [(3)H]norepinephrine ([(3)H]NE). The mechanism of inhibition was evaluated using Schild analysis. To eliminate the interaction of bupropion with DAT or NET, nomifensine or desipramine, respectively, was included in the superfusion buffer. A high bupropion concentration (100 microM) elicited intrinsic activity in the [(3)H]DA release assay. However, none of the concentrations (1 nM-100 microM) examined evoked [(3)H]NE overflow and, thus, were without intrinsic activity in this assay. Moreover, bupropion inhibited both nicotine-evoked [(3)H]DA overflow (IC(50) = 1.27 microM) and nicotine-evoked [(3)H]NE overflow (IC(50) = 323 nM) at bupropion concentrations well below those eliciting intrinsic activity. Results from Schild analyses suggest that bupropion competitively inhibits nicotine-evoked [(3)H]DA overflow, whereas evidence for receptor reserve was obtained upon assessment of bupropion inhibition of nicotine-evoked [(3)H]NE overflow. Thus, bupropion acts as an antagonist at alpha3beta2* and alpha3beta4* nAChRs in rat striatum and hippocampus, respectively, across the same concentration range that inhibits DAT and NET function. The combination of nAChR and transporter inhibition produced by bupropion may contribute to its clinical efficacy as a smoking cessation agent.     

Evaluation of the blood-brain barrier transport,population pharmacokinetics,and brain distribution of benztropine analogs and cocaine using in vitro and in vivo techniques

The N-substituted 3alpha-[bis(4'-fluorophenyl)methoxy]tropanes (AHN 2-003, AHN 1-055, AHN 2-005, and JHW 007) bind with high affinity to the dopamine transporter and inhibit dopamine uptake more potently than cocaine, but they demonstrate behavioral profiles in animal models of psychostimulant abuse that are unlike that of cocaine. The objective of this study was to characterize the in vitro permeability, brain distribution, and pharmacokinetics of the benztropine (BZT) analogs. Transport studies of cocaine and the BZT analogs (10-4 M) were conducted across bovine brain microvessel endothelial cells. Male Sprague-Dawley rats (approximately 300 g) were administered BZT analogs (10 mg/kg) or cocaine (5 mg/kg) via the tail vein. Blood and brain samples were collected over 36 h and assayed using UV-high-performance liquid chromatography. Transport of both AHN 1-055 (2.15 x 10-4 cm/s) and JHW 007 (2.83 x 10-4 cm/s) was higher (p < 0.05) than that of cocaine (1.63 x 10-4 cm/s). The volume of distribution (12.3-30.5 l/kg) of the analogs was significantly higher than cocaine (0.9 l/kg). The BZT analogs displayed a > or =8-fold higher elimination half-life (4.12-16.49 h) compared with cocaine (0.49 h). The brain-to-plasma partition coefficients were at least two-fold higher for the BZTs versus cocaine, except for AHN 2-003. The BZT analogs are highly permeable across the blood-brain barrier and possess a pharmacokinetic profile different from that of cocaine. These characteristics, in addition to their distinctive behavioral profiles, suggest that the BZT analogs may be promising candidates for the treatment of cocaine abuse.     

Behavioral effects of novel cocaine analogs: a comparison with in vivo receptor binding potency.

Several novel cocaine analogs, previously shown to be very potent in in vitro binding studies, have been examined for their stimulatory effects on locomotor activity and for their ability to displace [3H]WIN 35,428 binding in vivo in mice. These compounds, like WIN 35,428, lack an ester link between the phenyl group and the tropane ring and have para-substitutions on the phenyl ring. They were much more potent than (-)-cocaine in producing increases in locomotor activity. In addition, they were more potent than (-)-cocaine in inhibiting [3H]WIN 35,428 binding in vivo in mouse striatum. Thus, these compounds demonstrate similar high potency in behavioral tests and in receptor binding assays, both in vivo and in vitro. Results support the hypothesis of a relationship between binding at the dopamine transporter and the behavioral effects of cocaine-like drugs. Further, assuming that maximal occupancy occurs with total displacement of [3H]WIN 35,428 binding in vivo, the data suggest that maximal locomotor effects occur with near total occupancy of transporter binding sites.     

Anticonvulsant and adverse effects of avermectin analogs in mice are mediated through the gamma-aminobutyric acid(A) receptor

Twenty-five avermectin analogs were assessed in a mouse seizure model. The ED(50) against pentylenetetrazole-induced tonic seizures ranged from 0.48 mg/kg (L-676,893) to >160 mg/kg (L-685,869) cf. 0. 26 mg/kg for diazepam. Although avermectins are without acute toxic effects, they have been historically shown to have relative low LD(50) values in mammals. The mechanisms involved in the anticonvulsant effect and the toxicity were investigated. A series of avermectin analogs displaced [(3)H]ivermectin binding to rat brain membranes and recombinant GABA(A) receptors (alpha1beta3gamma2-subtype) with the same affinities, strongly suggesting that [(3)H]ivermectin labels the GABA(A) receptor in rodent brain. Avermectins, which were anticonvulsant, were also potent inhibitors of [(3)H]ivermectin binding in rat brain. However, the rank order for anticonvulsant activity did not parallel the rank order for affinity at the [(3)H]ivermectin site and it was reasoned that avermectins may have differential affinity or efficacy at subtypes of the GABA(A) receptor. All the active compounds tested potentiated the effects of GABA at recombinant GABA(A) receptors in oocytes and at native cortical GABA(A) receptors and the efficacy of avermectins at the GABA(A) receptor correlated best with their anticonvulsant potency. Although avermectins weakly inhibited [(3)H]strychnine binding in rat spinal cord, and inhibited glycine responses on primary cultured cortical neurons, activity at glycine receptors did not correlate with either anticonvulsant activity or toxicity. Because both anticonvulsant activity and toxicity correlated best with activity at GABA(A) receptors, it is unlikely that these effects can be separated, which may contraindicate the potential use of avermectins as anticonvulsants.     

The reinforcing efficacy of psychostimulants in rhesus monkeys: the role of pharmacokinetics and pharmacodynamics

This study was undertaken to investigate pharmacological variables that influence the reinforcing efficacy of psychostimulants. Rhesus monkeys (n = 9) responded under a within-session, exponentially increasing, progressive ratio schedule of cocaine reinforcement. Doses of cocaine, methylphenidate (MP), cocaine analogs [(+/-)-2beta-propanoyl-3beta-(2-naphthyl)-tropane (WF-23), HD-23; (+/-)-2beta-propanoyl-3beta-(2-isopropenyl)tropane (WF-60), HD-60; and 2beta-propanoyl-3beta-(4-tolyl)-tropane (HD-11, WF-11), and 2beta-propanoyl-3beta-(4-tolyl)-tropane (HD-11, WF-11), PTT], and MP analogs [(alphaR,2R)-alpha-(2-naphthalenyl)-2-piperidineacetic acid methyl ester, HDMP-28; and (alphaR,2S)-alpha-(2-naphthalenyl)-2-pyrrolideneacetic acid methyl ester, HDMP-29] that varied in their pharmacokinetic and pharmacodynamic properties were substituted for cocaine. These drugs were chosen according to their selectivity for dopamine transporters (DAT) and 5-hydroxytryptamine (serotonin) transporters (5-HTT) as assessed in rodents and their duration of action. In addition, data pertaining to the rate of onset at DAT were collected for the cocaine analogs using an ex vivo binding assay in rodent tissue. Finally, the pharmacodynamic profile of select drugs was confirmed in primate brain tissue. All drugs had reinforcing effects except HDMP-29. The rank ordering of the peak breaking points (BPs) was cocaine = MP = HDMP-28 >or= HD-60 >or= PTT >or= HD-23 > HDMP-29. The time to peak DAT occupancy for the cocaine analogs was greater than 30 min. The potency to maintain peak BP was significantly correlated with DAT affinity. There was not a linear relationship between monoamine transporter affinity and reinforcing efficacy, but it appeared that in nonhuman primates there is a range of DAT affinity under which maximal responding is maintained. Interestingly, the 5-HTT-selective cocaine analog HD-60 functioned robustly as a reinforcer at several doses in all monkeys tested. These data question the dogma regarding the role of pharmacokinetic factors and the relative influence of DAT and 5-HTT in stimulant reinforcement.     

Reinforcing strength of a novel dopamine transporter ligand: pharmacodynamic and pharmacokinetic mechanisms

Drugs that block dopamine uptake often function as positive reinforcers but can differ along the dimension of strength or effectiveness as a positive reinforcer. The present study was designed to examine pharmacological mechanisms that might contribute to differences in reinforcing strength between the piperidine-based cocaine analog (+)-methyl 4beta-(4-chlorophenyl)-1-methylpiperidine-3-alpha-carboxylate [(+)-CPCA] and cocaine. Drugs were made available to rhesus monkeys (n = 5) for i.v. self-administration under a progressive ratio schedule. Both compounds maintained responding with sigmoidal or biphasic dose-response functions (0.1-1.0 mg/kg/injection). (+)-CPCA was one-fourth as potent as cocaine and maintained fewer injections per session, at maximum. For in vitro binding in monkey brain tissue, (+)-CPCA was about one-half as potent as cocaine at the dopamine transporter (DAT), and the two compounds had similar affinities at the norepinephrine transporter. (+)-CPCA was less than 1/10 as potent as cocaine at the serotonin transporter. In ex vivo binding in rat striatum, occupancy of the DAT increased directly with dose to a maximum of approximately 80% for both compounds, and (+)-CPCA was about one-fourth as potent as cocaine. Ex vivo DAT occupancy was significantly higher for cocaine than (+)-CPCA at 2 min after injection but similar at other times. Thus, the primary differences between these compounds were in serotonin transporter affinity and the kinetics of DAT binding. These results suggest that (+)-CPCA is a weaker positive reinforcer than cocaine because it has a slower onset of action over the first few minutes after i.v. injection.     

N,N'-Alkane-diyl-bis-3-picoliniums as nicotinic receptor antagonists: inhibition of nicotine-evoked dopamine release and hyperactivity

The current study evaluated a new series of N,N'-alkane-diyl-bis-3-picolinium (bAPi) analogs with C6-C12 methylene linkers as nicotinic acetylcholine receptor (nAChR) antagonists, for nicotine-evoked [3H]dopamine (DA) overflow, for blood-brain barrier choline transporter affinity, and for attenuation of discriminative stimulus and locomotor stimulant effects of nicotine. bAPi analogs exhibited little affinity for alpha4beta2* (* indicates putative nAChR subtype assignment) and alpha7* high-affinity ligand binding sites and exhibited no inhibition of DA transporter function. With the exception of C6, all analogs inhibited nicotine-evoked [3H]DA overflow (IC50 = 2 nM-6 microM; Imax = 54-64%), with N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB; C12) being most potent. bPiDDB did not inhibit electrically evoked [3H]DA overflow, suggesting specific nAChR inhibitory effects and a lack of toxicity to DA neurons. Schild analysis suggested that bPiDDB interacts in an orthosteric manner at nAChRs mediating nicotine-evoked [3H]DA overflow. To determine whether bPiDDB interacts with alpha-conotoxin MII-sensitive alpha6beta2-containing nAChRs, slices were exposed concomitantly to maximally effective concentrations of bPiDDB (10 nM) and alpha-conotoxin MII (1 nM). Inhibition of nicotine-evoked [3H]DA overflow was not different with the combination compared with either antagonist alone, suggesting that bPiDDB interacts with alpha6beta2-containing nAChRs. C7, C8, C10, and C12 analogs exhibited high affinity for the blood-brain barrier choline transporter in vivo, suggesting brain bioavailability. Although none of the analogs altered the discriminative stimulus effect of nicotine, C8, C9, C10, and C12 analogs decreased nicotine-induced hyperactivity in nicotine-sensitized rats, without reducing spontaneous activity. Further development of nAChR antagonists that inhibit nicotine-evoked DA release and penetrate brain to antagonize DA-mediated locomotor stimulant effects of nicotine as novel treatments for nicotine addiction is warranted.