Effects of dopamine transporter selective 3-phenyltropane analogs on locomotor activity, drug discrimination, and cocaine self-administration after oral administration

European Journal of Pharmacology 553 (2006) 149 – 156 www.elsevier.com/locate/ejphar

Effects of dopamine transporter selective 3-phenyltropane analogs on locomotor activity, drug discrimination, and cocaine self-administration after oral administration F. Ivy Carroll a,⁎, Barbara S. Fox b , Michael J. Kuhar c , James L. Howard d , Gerald T. Pollard d , Susan Schenk e a

Center for Organic and Medicinal Chemistry, Research Triangle Institute, Research Triangle Park, NC 27709, USA b Addiction Therapies, Inc., Wayland, MA 01778, USA c Yerkes National Primate Research Center of Emory University, Atlanta, GA 30329, USA d Howard Associates, LLC, Research Triangle Park, NC 27709, USA e Victoria University of Wellington, School of Psychology, Wellington, New Zealand Received 12 June 2006; received in revised form 7 September 2006; accepted 11 September 2006 Available online 23 September 2006

Abstract Several studies suggest that a dopamine transporter uptake inhibitor that has a slower onset and longer duration of action than cocaine in animal behavioral measures and decreases cocaine self-administration would be useful as an indirect dopamine agonist pharmacotherapy to treat cocaine addiction. In the present study, we compared five 3-phenyltropane analogs administered orally in locomotor activity in mice and drug discrimination in rats to gain information concerning relative potency, onset, and duration of action. The compounds were also evaluated for reduction of cocaine self-administration in rats after oral administration. In general, the compounds had a slower onset of action than cocaine and reduced cocaine selfadministration. 3β-(4-Chlorophenyl)-2β-(3-(4′-methylphenyl)-isoxazol-5-yl)tropane (RTI-336) was the most potent in locomotor activity and drug discrimination; it was less stimulatory than cocaine in the first hour and had the slowest onset and longest duration of action. It also reduced selfadministration of two infusion doses of cocaine in the rat. © 2006 Elsevier B.V. All rights reserved. Keywords: 3-Phenyltropane; Locomotor activity; Drug discrimination; Self-administration; Dopamine transporter

1. Introduction There are currently no medications available for effectively treating cocaine addiction. One possible approach to the development of such an agent would be the selection of an indirect dopamine agonist. Some of the most effective pharmacotherapies for substance abuse are substitute agonists, most notably nicotine replacement therapy as an aid for smoking cessation and methadone as a treatment for heroin addiction. An indirect dopamine agonist for cocaine should possess some but not all of cocaine's attributes. The reinforcing effects of cocaine appear related to its action at the dopamine transporter (DAT). Compounds that inhibit the DAT ⁎ Corresponding author. Tel.: +1 919 541 6679; fax: +1 919 541 8868. E-mail address: [email protected] (F.I. Carroll). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.09.024

function are positive reinforcers. Their potency in self-administration studies is positively correlated with their binding affinity at the DAT (Bergman et al., 1989; Ritz et al., 1987; Wee et al., 2006; Wilcox et al., 2000; Woolverton and Wang, 2004). However, potency as a reinforcer is not the only factor involved. In fact, cocaine has a relatively weak affinity for the DAT and low potency as a reinforcer but is one of the most efficacious reinforcers in animals and is highly abused by humans. Thus, factors other than binding affinity appear to contribute to reinforcing efficacy. The mode of cocaine administration can significantly influence the euphoric experience, presumably as a result of varied pharmacokinetics. The low potency, yet high efficacy of cocaine as a reinforcer, has been attributed to its fast onset of action (Fowler et al., 1998; Stathis et al., 1995; Volkow et al., 2003). A recent report suggests that higher rates of drug administration may support addiction by more extensively activating

150

F.I. Carroll et al. / European Journal of Pharmacology 553 (2006) 149–156

mesocorticolimbic circuitry and inducing behavior plasticity (Samaha and Robinson, 2005). In addition to rate of onset, the duration of action of a drug may influence reinforcement efficacy. Volkow and coworkers (Volkow et al., 2003) used positron emission tomography (PET) to show that the rate of clearance for the relatively more potent methylphenidate was significantly slower than for cocaine and suggested that this could account for the much lesser abuse of methylphenidate than cocaine despite their otherwise similar pharmacological properties. Thus, different pharmacokinetic parameters, including rate of onset and duration of action, are likely to influence the rewarding value and contribute to the ability to induce neuroplasticity associated with addiction. Compounds selective for the DAT relative to the norepinephrine transporter (NET) and serotonin transporter (5-HTT) are of particular interest as potential pharmacotherapies to treat cocaine addiction (Grabowski et al., 2004). We developed a number of 3phenyltropane analogs that are selective for the DAT relative to the 5-HTT and NET in inhibition of radioligand binding (Carroll et al., 1995, 2004). Only a few of the compounds have been evaluated in behavioral pharmacology tests, and even fewer have been tested after oral administration. In this study, we report that five previously reported DAT selective 3-phenyltropane analogs (Carroll et al., 1995, 2004) are also relatively selective for a number of other receptor enzymes and ion channels. In addition, we show that the compounds increase locomotor activity, generalize to cocaine in a discrimination test, and reduce cocaine selfadministration in a rat model after oral administration. Some of the compounds show slow onset and a long duration of action relative to cocaine, with locomotor stimulation less than cocaine's, properties needed for an indirect dopamine agonist pharmacotherapy for cocaine addiction. 2. Materials and methods All animals received care according to “Guide for the Care and Use of Laboratory Animals,” United States Department of Health and Human Services Publication, Revised, 1996. The animal care facilities were certified by the American Association for Accreditation of Laboratory Animal Care. These studies were approved by the Institutional Animal Care and Use Committees at the Research Triangle Institute, Victoria University of Wellington, or the University of North Texas Health Science Center. Housing and experimental conditions were as nearly identical as possible at the three institutions. 2.1. Inhibition of monoamine transporter binding by 3-phenyltropane analogs Brains from male Sprague Dawley albino rats weighing 200– 250 g (Harlan-Sprague Dawley, Indianapolis, IN, USA) were removed, dissected, and rapidly frozen. Ligand binding experiments for the DATwere conducted in assay tubes containing 0.5 ml buffer (10 mM sodium phosphate containing 0.32 M sucrose, pH 7.4) on ice for 120 min. Each assay tube contained 0.5 nM [3H]3β-(4-fluorophenyl)-2β-tropanecarboxylic acid methyl ester ([3H] WIN 35,428) and 0.1 mg striatal tissue (original wet weight). The

nonspecific binding of [3H]WIN 35,428 was defined using 30 μM (-)-cocaine. Ligand binding experiments for the 5-HTT were conducted in assay tubes containing 4 ml of buffer (50 mM Tris, 120 mM NaC1, 5 mM KC1, pH 7.4 at 25 °C) for 90 min at room temperature. Each assay tube contained 0.2 nM [3H]paroxetine and 1.5 mg of midbrain tissue (original wet weight). Nonspecific binding of [3H]paroxetine was defined by 1 μM citalopram. Ligand binding experiments for the NET were conducted in Tris buffer (50 mM Tris, 120 mM NaC1, 5 mM KC1, pH 7.4 at 4 °C) at a total volume of 0.5 ml. Each assay tube contained 0.5 nM [3H] nisoxetine and 8 mg of rat cerebral cortex. The nonspecific binding of [3H]nisoxetine was defined using 1 μM desipramine. Incubations were terminated by filtration with three 5-ml washes of icecold buffer through GF/B filters that were previously soaked in 0.05% polyethylenimine. Results were analyzed using the Equilibrium Binding Data Analysis software (EBDA, Biosoft). 2.2. NovaScreen The five 3-phenyltropane analogs were evaluated at 10,000 nM for inhibition of binding in a 62-assay NovaScreen (Biosciences Corp., Hanover, MO, USA). An estimated Ki was determined for each assay that showed greater than 50% inhibition. 2.3. Locomotor activity 2.3.1. Subjects Male CD-1 mice, 19–28 g (Charles River Laboratories, Raleigh, NC, USA), were habituated to the Animal Research Facility for at least 5 days. 2.3.2. Apparatus and procedure Activity was measured in 24 plexiglass chambers, 16″ × 8″ × 8″, each set in an array of 4 photocells in a Cage Rack system (San Diego Instruments, San Diego, CA, USA). Doses were selected on the basis of previous i.p. results. Compounds were prepared by homogenizing in 0.5% methyl cellulose and were dosed at 3, 10, or 30 mg/kg in a volume of 10 ml/kg of body weight. Mice (N = 5 or 6 per dose and vehicle) were habituated to the activity chambers for 1 / 2 h, then removed individually, dosed p.o., and replaced. Photobeam interruptions were recorded in 10-min bins for 4 h. 2.3.3. Analyses Data were grouped into 1-h time bins and subjected to analysis of variance, with the Newman-Keuls test applied post hoc at each time point where a main effect of dose or a dose x time interaction was significant (P b 0.05). An ED50 was determined for the hour showing the greatest change from control by using a sigmoidal dose-response (variable slope) curve fitting procedure (GraphPad Prism, GraphPad Software, Inc., San Diego, CA, USA). 2.4. Drug discrimination 2.4.1. Subjects Adult male rats (Sprague Dawley albino) from HarlanSprague Dawley (Indianapolis, IN, USA) were maintained one per cage on a 12/12-h light/dark cycle (lights on 07.00 to 19.00)

F.I. Carroll et al. / European Journal of Pharmacology 553 (2006) 149–156

in an animal housing room with controlled temperature (22 °C) and humidity (40–60%). Body weights were maintained at 320–350 g by limiting food intake to 20 g/day including the food earned during the operant session. 2.4.2. Apparatus and procedure Training and testing were done in standard rat operant chambers inside sound-attenuating enclosures (Coulbourn Instruments, Allentown, PA, USA). Each chamber was equipped with a house light, two response levers, a food trough between the levers, and a dispenser for 45-mg food pellets (BioServ, Frenchtown, NJ, USA). Programming of contingencies and data acquisition was done by IBM-PC compatible computers via LVB interfaces (Med Associates, East Fairfield, VT, USA). 2.4.3. Cocaine discrimination training Rats were trained to discriminate cocaine from saline in daily sessions 5 days/week. Ten minutes before each session, either cocaine 10 mg/kg or saline vehicle was dosed i.p. in a volume of 1 ml/kg of body weight. For half of the subjects, after cocaine injection, every 10 presses on the left lever delivered a pellet, and presses on the right lever had no programmed consequence; after saline injection, every 10 presses on the right lever delivered a pellet, and presses on the left lever had no programmed consequence. For the other half of the subjects, this contingency was reversed. A correct lever choice for a session was defined as earning the first pellet with 12 or fewer presses (i.e., no more than 2 on the incorrect lever). Cocaine (C) or saline (S) was assigned such that each was given on 2 consecutive days (e.g., for the 5 days of 1 week, C–C–S–S–C, and for the next week, C–S–S–C–C). Sessions were terminated after rats earned 20 pellets or 10 min elapsed. The criterion for stability on the discrimination was 10 consecutive sessions in which rats chose the correct lever on the first FR-10. To maintain stability, a subject had to have made the correct choice in the most recent drug-lever-correct and the most recent salinelever-correct sessions. 2.4.4. Compound testing for generalization with the cocaine cue Test sessions lasted for a maximum of 20 min, and both levers were active; 10 presses on either lever led to reinforcement. Days were assigned to cocaine, saline, and test compound in a double alternation sequence such that a test day occurred 3 times every 2 weeks, with sequential test days separated by at least 1 cocaine and 1 saline day. A compound was tested initially at doses that produced stimulation in mouse locomotor activity, and then a full dose-effect curve from 20% or less to 80% or greater generalization at 45 min was generated. The highest dose was that which reduced lever pressing by N50%, or produced significant side effects, or 50 mg/kg, whichever was lower. Degree of generalization was defined as the percentage of subjects that chose the cocaine-appropriate lever. Doses of a compound were tested in 3 or 6 subjects. Compounds were prepared in 2% methyl cellulose and were dosed p.o. in a volume of 1 ml/kg 45, 90, 180, or 360 min before the session.

151

2.4.5. Analyses Lever choice on a test day was defined as the first lever on which 10 presses occurred. Total presses for the session also were recorded. Results for a compound were tabulated as the percent of subjects choosing the cocaine lever. Where cocainelever choice reached 75%, an ED50 at the 45-min time point was calculated with GraphPad Prism. 2.5. Cocaine self-administration 2.5.1. Subjects Subjects were male Sprague Dawley albino rats weighing 350–400 g (bred in the vivarium at Victoria University of Wellington, New Zealand). They were housed individually in hanging polycarbonate cages in a temperature- (21 °C) and humidity-controlled (70%) animal colony. Lights were on at 7.5 h and off at 20.5 h. All behavioral testing was carried out during the light portion of the cycle. Food and tap water were freely available except during testing. 2.5.2. Apparatus Each of 24 self-administration chambers (Med-Associates, ENV-001) was equipped with two levers. Depression of one lever (the “active” lever) resulted in the delivery of an intravenous infusion of cocaine hydrochloride. Coincident with drug delivery was the illumination of a cue lamp located directly above the lever. Depression of the other lever (the “inactive” lever) was without programmed consequence. Drug delivery was via motorized pumps (Razel, model A with 1-rpm motors) and was controlled by IBM-compatible computers interfaced with the operant chambers through the OPN software package (Spencer and Emmett-Oglesby, 1985). 2.5.3. Surgery Deep anesthesia was produced by separate injections of ketamine (60 mg/kg i.p.) and sodium pentobarbital (20 mg/kg i. p.). A silastic cannula was inserted into the external jugular vein, and the distal end was passed subcutaneously to an exposed portion of the skull, where it was affixed with dental acrylic to four stainless-steel screws embedded in the skull. Five to 7 days were allowed for recovery. On each of these days, the cannulae were flushed with 0.1 ml of a sterile saline solution containing heparin (30 U/ml) and penicillin G potassium (250,000 U/ml) to maintain patency. On self-administration test days, the cannulae were flushed with this solution both prior to and following the test. 2.5.4. Training During an initial acquisition phase, the rats were exposed to the test chamber for daily 2-h self-administration sessions. On these days, depression of the active lever resulted in the delivery of cocaine (0.5 mg/kg/infusion) on an FR-1 schedule of reinforcement. Following acquisition, the ratio requirements were increased so that the cocaine reinforcer was delivered after completion of 5 active lever responses (FR-5). Effects of the five 3-phenyltropane analogs were evaluated against self-administration of 0.5 mg/kg/ infusion cocaine for some rats and 0.25 mg/kg/infusion for others.

152

F.I. Carroll et al. / European Journal of Pharmacology 553 (2006) 149–156

2.5.5. Drug testing Test sessions were conducted daily. Baseline (vehicle gavage) tests were conducted on at least two test sessions prior to test compound evaluation, and the resulting data were averaged for each subject to produce data for the 0.0 dosage condition. Each compound, suspended in 1% Tween 80, was administered via oral gavage in a volume of 4.0 ml/kg 15 min prior to the self-administration tests. 2.5.6. Analyses The measure of interest was the number of infusions of cocaine self-administered by the rat after vehicle and the three dose levels of test article. These values were submitted to analysis of variance. An effect was considered significant if P b 0.05. 2.6. Drugs The 3-phenyltropane analogs, 3β-(4-methylphenyl)tropane2β-carboxylic acid isopropyl ester (RTI-117), 3β-(4-methylphenyl)-2β-(3-phenylisoxazol-5yl)tropane (RTI-176), 3β-(4chlorophenyl)-2β-(3-(3-methylphenyl)isoxazol-5-yl)tropane (RTI-336), 3β-(4-methylphenyl)-2β-(3-ethylisoxazol-5-yl)tropane (RTI-354), and 3β-(4-methylphenyl)-2β-(3-(4-methoxyphenyl)-isoxazol-5-yl)tropane (RTI-386), were synthesized as previously reported and were greater than 98% pure (Carroll et al., 1995, 2004). The structures are shown in Fig. 1. Cocaine was supplied by the National Institute on Drug Abuse Drug Supply Program. 3. Results 3.1. Inhibition of radioligand binding at monoamine transporters All five 3-phenyltropane analogs showed relatively high affinity for inhibition of [3H]WIN 35,428 binding to rat brain DAT. The IC50s ranged from 1.58 to 6.45 nM (Table 1). Thus, these affinities are 4- to 15-fold greater than that of the lead compound 3β-phenyltropane-2β-carboxylic acid methyl ester (WIN 35,065-2) and 14- to 56-fold greater than that of cocaine. The affinities for inhibition of binding at the NET and 5-HTT were much higher, with Kis of 180 to 1160 nM and 366 to 582 nM, respectively. RTI-176 and RTI-354 with IC50s of 1.58 and 1.62 nM at the DAT were slightly more potent than RTI-336 and RTI-386, which have IC50s of 4.09 and 3.93 nM at the DAT.

Fig. 1. Structure of RTI-117, -176, -336, -354, and -386.

Table 1 Affinities of 3-phenyltropane analogs for inhibition of binding of the DAT, NET, and 5-HTT Compound

DAT, IC50 (nM) [3H] WIN 35,428

NET, Ki (nM) [3H] Nisoxetine

5-HTT, Ki (nM) [3H]Paroxetine

RTI-117a RTI-176b RTI-336b RTI-354b RTI-386b WIN 35,065-2b Cocainea

6.45 ± 0.85 1.58 ± 0.02 4.09 ± 0.44 1.62 ± 1.0 3.93 ± 0.49 23 ± 5

1160 ± 23 239 ± 11 1030 ± 24 180 ± 13 455 ± 89 550 ± 44

554 ± 44 465 ± 48 522 ± 48 582 ± 53 366 ± 2.9 178 ± 5.5

89 ± 4.8

1900 ± 170

95 ± 8

a

Data taken from Carroll et al. (1995). bData taken from Carroll et al. (2004).

All five compounds have greater than 100-fold selectivity for the DAT relative to the NET and 5-HTT, whereas cocaine is about equipotent. 3.2. Inhibition of binding at 62 different receptors, enzymes, and ion channels None of the 3-phenyltropane analogs had appreciable affinity in any of the 62 assays tested. The results are summarized in Table 2. RTI-117, -176, -336, -354, and -386 showed greater than 50% inhibition at the 10,000 nM concentration in 6, 1, 7, 0, and 6 assays, respectively. RTI-117 had its greatest affinity at the central muscarinic receptor, with a Ki of 1530 nM, which is 237fold larger than its affinity at the DAT. RTI-176 had a Ki of 8175 nM at sodium channel site 2, which is over 5000-fold greater than its DAT affinity. RTI-336 was 91-fold selective for DAT relative to the M1 human recombinant muscarinic receptor, where it shows its highest affinity. RTI-386 showed highest affinity at the cannabinoid CB2 receptor, with a Ki of 1115 nM, but is 285-fold selective for the DAT relative to this receptor. 3.3. Locomotor activity The compounds produced a dose-related increase in locomotor activity in mice after p.o. administration (Table 3). The ED50s were almost identical in their hour of peak activity, Table 2 Estimated Kis (nM) of DAT selective 3-phenyltropanes in NovaScreen assays Assay

RTI-117 RTI-176 RTI-336 RTI-354 RTI-386

Adrenergic, alpha1 non5110 selective Adrenergic, alpha1A 14,600 Adrenergic, alpha1 1B 2475 Muscarinic M1HR a Muscarinic M2HR a Muscarinic, non-selective, 1530 central Muscarinic, non-selective, 5980 peripheral Sodium, site 2 Cannabinoid, CB1, HR a Cannabinoid, CB2, HR a Calcium channel, Type L a

HR = human recombinant.

8175

372 1410 428

2320 2815

975

2850

64,900 28,250 3000

4320 1115 7445

F.I. Carroll et al. / European Journal of Pharmacology 553 (2006) 149–156

153

Table 3 Percent change from vehicle in locomotor activity in mice for 3-phenyltropane analogs (p.o.)

a Route of administration of cocaine was i.p. The data are reproduced from Carroll et al. (2004). bDifferent from vehicle by Newman-Keuls following analysis of variance, P b 0.05. Boxed values represent the hour of greatest percent change in activity.

ranging from 10.0 to 16.1 mg/kg. Four of the compounds, RTI117, -176, -336, and -386 were less stimulating than cocaine in the first hour, with maximums ranging from 44 to 77% of that of cocaine. RTI-354 showed activity 67% greater than cocaine. The peak time of activity varied from the first hour to the fourth hour, with RTI-336 showing the least onset in the first hour and being the only compound in which the first hour activity was not significant. 3.4. Drug discrimination All five compounds showed generalization to cocaine after p.o. administration (Table 4). The ED50s varied from 5.8 to 36.7 mg/kg, with RTI-336 being the most potent and RTI-386 the least potent. RTI-117 and -354 showed greater than 80% generalization only at the 45-min time point. RTI-176 showed greater than 80% generalization at both 45- and 90-min time points. RTI-336 and -386 showed full generalization up to 180 min.

following: for RTI-117 there was an effect of dose on the 0.5 mg/ kg/infusion of cocaine [F(3,15) = 10.366, P b 0.01] and the 0.25 mg/kg/infusion [F(3,12) = 3.965, P b 0.05]. For RTI-176 there was a trend on the 0.5 mg/kg/infusion [F(3,12) = 1.571, ns] Table 4 Drug discrimination effects in rats (p.o.) Percent of rats choosing the cocaine lever at dose (mg/kg) Compound

Pretreatment time

RTI-117

45 90 180 360

0 0 0 0

0 0 0 0

33 33 0 0

100 67 0 0

RTI-176

45 90 180 360

33 0 0 33

0 33 33 0

17 0 0 0

50 33 0 0

RTI-336

45 90 180 360

0 33 0 0

17 33 33 33

100 100 100 67

RTI-354

45 90 180 360

0 33 0 0

33 17 0 17

50 0 0 0

83 67 67 33

RTI-386

45 90 180 360

17 0 0 0

17 0 0 0

0 0 0 0

0 33 33 0

3.5. Effects on cocaine self-administration With the exception of RTI-176 at the 0.5 mg/kg/infusion of cocaine, all five compounds reduced cocaine self-administration at both the 0.25 and 0.5 mg/kg/infusions. Figs. 2–6 show average active lever responding maintained by 0.25 mg/kg/infusion (left panel) or 0.5 mg/kg/infusion (right panel) of cocaine and the effects thereon of the five compounds. Responding maintained by cocaine was dose-dependent, and more responses were produced when the lower dose of cocaine was available for selfadministration. An ANOVA for each compound revealed the

2.5

5

10

25

50

ED50 mg/kg 11.5

83 83 33 0

21.9

5.8

8.6

83 67 83 17

36.7

154

F.I. Carroll et al. / European Journal of Pharmacology 553 (2006) 149–156

Fig. 2. Effects of RTI-117 on cocaine self-administration. ANOVA indicated effects of RTI-117 on responding maintained by both doses of cocaine and Tukey post-hoc tests indicated significant differences from vehicle following administration of the highest dose (* = P b 0.05).

Fig. 3. Effects of RTI-176 on cocaine self-administration. ANOVA indicated effects of RTI-176 on responding maintained by 0.25 mg/kg/infusion cocaine and Tukey post-hoc tests indicated significant differences from vehicle following administration of the highest dose (* = P b 0.05).

Fig. 4. Effects of RTI-336 on cocaine self-administration. ANOVA indicated effects of RTI-336 on responding maintained by both doses of cocaine and Tukey posthoc tests indicated significant differences from vehicle following administration of the highest dose (* = P b 0.05).

Fig. 5. Effects of RTI-354 on cocaine self-administration. ANOVA indicated effects of RTI-354 on responding maintained by both doses of cocaine and Tukey posthoc tests indicated significant differences from vehicle following administration of the highest dose (* = P b 0.05).

F.I. Carroll et al. / European Journal of Pharmacology 553 (2006) 149–156

155

Fig. 6. Effects of RTI-386 on cocaine self-administration. ANOVA indicated effects of RTI-386 on responding maintained by both doses of cocaine and Tukey posthoc tests indicated significant differences from vehicle following administration of the highest dose (* = P b 0.05).

and an effect on the 0.25 mg/kg/infusion [F(3,12) = 4.367, P b 0.05]. For RTI-336 there was an effect on the 0.5 mg/kg/ infusion [F(3,12) = 11.382, P b 0.01] and the 0.25 mg/kg/infusion [F(3,12) = 29.248, P b 0.05]. For RTI-354 there was an effect on the 0.5 mg/kg/infusion [F(3,12) = 5.796, P b 0.05] and the 0.25 mg/kg/infusion [F(3,12) = 17.347, P b 0.01]. For RTI-386 there was an effect on the 0.5 mg/kg/infusion [F(91,4) = 91.38, P b 0.01] and the 0.25 mg/kg/infusion [F(1,4) = 21.81, P = 0.01]. 4. Discussion The lead compound for the 3-phenyltropane class of DAT uptake inhibitors is WIN 35,065-2. In our studies, it has 3.8 and 3.5 times higher affinity than cocaine at the DAT and NET, respectively, and is 1.9 times less potent at the 5-HTT. All five of the new 3-phenyltropanes have higher affinity at the DAT than WIN 35,065-2 and are selective for the DAT relative to the NET and 5-HTT. In addition, all five compounds are selective for the DAT relative to 62 receptor, enzyme, and ion channel assays determined in a NovaScreen (Biosciences Corp., Hanover, MO, USA). In general, it is thought that the faster a drug reaches the brain and exerts its psychoactive effects, the greater its reinforcing effects on abuse liability (Nelson et al., 2006; Stathis et al., 1995). It is interesting to note that like cocaine, RTI-117, -176, -354, and -386 all show locomotor activity different from vehicle for the first hour. In contrast, RTI-336 did not show locomotor activity different from vehicle until the second hour. With the exception of RTI-354, the maximal effects in the first hour were 44 to 77% of that produced by cocaine. PET imaging studies have shown that drugs that have longer durations of action are less abused than drugs like cocaine, which have a short duration of action (Volkow et al., 2003). In this study, cocaine and RTI-117 had their peak locomotor activity stimulant effect in the first hour, RTI-354 in the second hour, RTI-176 in the third hour, and RTI-336 and RTI-386 in the fourth hour, which was the longest duration tested. In the drug discrimination test, RTI-336 had the longest duration of action, showing full generalization up to 180 min and partial generation even at 360 min. RTI-336, with an ED50 of 5.8 mg/kg, was also the most potent analog in the drug discrimination test. The longer duration of action of the 3-phenyltropanes relative to cocaine probably depends on several factors but in part is

likely due to the greater chemical and enzymatic stability of the 3-phenyltropanes relative to cocaine. In contrast to cocaine, which has ester groups at both the 2- and 3-positions on the tropane ring, all five 3-phenyltropanes have a stable substituted phenyl ring connected to the 3-position on the tropane ring. In addition, with the exception of RTI-117, the 3-phenyltropanes have a chemically stable 3-substituted isoxazol ring connected to the 2β-position on the tropane ring. RTI-117, which had the shortest duration of action of all the 3-phenyltropanes, has an ester group at the 2-position. Pretreatment with four of the selective dopamine uptake inhibitors, RTI-117, -336, -354, and -386, decreased responding maintained by two different cocaine infusion doses. The decrease for RTI-117 was not as impressive as that for the other three compounds. RTI-117 has a shorter duration of action in both locomotor and drug discrimination than the other three 3phenyltropanes. Thus, the difference between RTI-117 and the three other compounds could be due to its shorter duration of action. RTI-176 decreased cocaine self-administration at 0.25 mg/kg/infusion but not at the 0.5 mg/kg/infusion at the doses tested. Since RTI-176 did not show generalization to cocaine in the drug discrimination assay until a dose of 50 mg/kg, it is possible that a higher dose of RTI-176 would have resulted in a more impressive decrease in responding. A 50-mg/kg dose of RTI-386 was also required to show generalization to cocaine. This compound showed a decrease in responding only at the 104.4-mg/kg dose, which is more than twice the highest dose used for RTI-176. The reduction of cocaine self-administration by all five of these DAT selective uptake inhibitors is consistent with previous studies, which showed that the DAT selective RTI113 and GBR 12,909 (vanoxerine) decreased cocaine selfadministration (Dworkin et al., 1998; Schenk, 2002). Blockade of the DAT by cocaine leads to increased extracellular DA, which then leads to increased DAT activity (Fleckenstein et al., 1999; Mash et al., 2002). Drug dependence might result from a deficit in the extracellular dopamine (DA) that results after cocaine is no longer present. A drug that normalized this DA deficit might be a good pharmacotherapy for cocaine dependence. Regardless of the mechanism, by analogy to methadone and buprenorphine as treatment for opiate abuse and nicotine for smokers, these 3-phenyltropane analogs may be useful as indirect dopamine agonist treatments for cocaine addiction. In general, these compounds produced less stimulation in

156

F.I. Carroll et al. / European Journal of Pharmacology 553 (2006) 149–156

the first hour and had slower onset of action than cocaine, and they reduced cocaine self-administration. In addition, they were active by oral administration, and both their stimulant and discriminative effects were long lasting. Based on these results, RTI-336 has the most favorable profile by virtue of its potency, slow onset, and long duration of action. Acknowledgement This work was supported by the United States National Institute on Drug Abuse (NIDA) Grant DA 05477, DA 11530, DA 13326, and DA 00418. We thank NIDA (CTDP, Division of Pharmacotherapies and Medical Consequences of Drug Abuse) for the drug discrimination data and for obtaining the NovaScreen assays. References Bergman, J., Madras, B.K., Johnson, S.E., Spealman, R.D., 1989. Effects of cocaine and related drugs in nonhuman primates. III. Self-administration by squirrel monkeys. J. Pharmacol. Exp. Ther. 251, 150–155. Carroll, F.I., Kotian, P., Dehghani, A., Gray, J.L., Kuzemko, M.A., Parham, K.A., Abraham, P., Lewin, A.H., Boja, J.W., Kuhar, M.J., 1995. Cocaine and 3β-(4′-substituted phenyl)tropane-2β-carboxylic acid ester and amide analogues. New high-affinity and selective compounds for the dopamine transporter. J. Med. Chem. 38, 379–388. Carroll, F.I., Pawlush, N., Kuhar, M.J., Pollard, G.T., Howard, J.L., 2004. Synthesis, monoamine transporter binding properties, and behavioral pharmacology of a series of 3β-(substituted phenyl)-2β-(3′-substituted isoxazol-5-yl)tropanes. J. Med. Chem. 47, 296–302. Dworkin, S.I., Lambert, P., Sizemore, G.M., Carroll, F.I., Kuhar, M.J., 1998. RTI-113 administration reduces cocaine self-administration at high occupancy of dopamine transporter. Synapse 30, 49–55. Fleckenstein, A.E., Haughey, H.M., Metzger, R.R., Kokoshka, J.M., Riddle, E.L., Hanson, J.E., Gibb, J.W., Hanson, G.R., 1999. Differential effects of psychostimulants and related agents on dopaminergic and serotonergic transporter function. Eur. J. Pharmacol. 382, 45–49.

Fowler, J.S., Volkow, N.D., Logan, J., Gatley, S.J., Pappas, N., King, P., Ding, Y.S., Wang, G.J., 1998. Measuring dopamine transporter occupancy by cocaine in vivo: radiotracer considerations. Synapse 28, 111–116. Grabowski, J., Shearer, J., Merrill, J., Negus, S.S., 2004. Agonist-like, replacement pharmacotherapy for stimulant abuse and dependence. Addict. Behav. 29, 1439–1464. Mash, D.C., Pablo, J., Ouyang, Q., Hearn, W.L., Izenwasser, S., 2002. Dopamine transport function is elevated in cocaine users. J. Neurochem. 81, 292–300. Nelson, R.A., Boyd, S.J., Ziegelstein, R.C., Herning, R., Cadet, J.L., Henningfield, J.E., Schuster, C.R., Contoreggi, C., Gorelick, D.A., 2006. Effect of rate of administration on subjective and physiological effects of intravenous cocaine in humans. Drug Alcohol Depend. 82, 19–24. Ritz, M.C., Lamb, R.J., Goldberg, S.R., Kuhar, M.J., 1987. Cocaine receptors on dopamine transporters are related to self-administration of cocaine. Science 237, 1219–1223. Samaha, A.N., Robinson, T.E., 2005. Why does the rapid delivery of drugs to the brain promote addiction? Trends Pharmacol. Sci. 26, 82–87. Schenk, S., 2002. Effects of GBR 12909, WIN 35,428 and indatraline on cocaine self-administration and cocaine seeking in rats. Psychopharmacology (Berl.) 160, 263–270. Spencer, D.G., Emmett-Oglesby, M.W., 1985. Parallel processing strategies in the application of microcomputers to the behavioral laboratory. Behav. Res. Meth. Instrum. 17, 294–300. Stathis, M., Scheffel, U., Lever, S.Z., Boja, J.W., Carroll, F.I., Kuhar, M.J., 1995. Rate of binding of various inhibitors at the dopamine transporter in vivo. Psychopharmacology (Berl.) 119, 376–384. Volkow, N.D., Fowler, J.S., Wang, G.-J., Shreeve, W.W., 2003. Imaging studies in substance abuse. In: Feinendegen, L.E. (Ed.), Molecular Nuclear Medicine. Springer-Verlag, Berlin, Germany. Wee, S., Carroll, F.I., Woolverton, W.L., 2006. A reduced rate of in vivo dopamine transporter binding is associated with lower relative reinforcing efficacy of stimulants. Neuropsychopharmacology 31, 351–362. Wilcox, K.M., Rowlett, J.K., Paul, I.A., Ordway, G.A., Woolverton, W.L., 2000. On the relationship between the dopamine transporter and the reinforcing effects of local anesthetics in rhesus monkeys: practical and theoretical concerns. Psychopharmacology (Berl.) 153, 139–147. Woolverton, W.L., Wang, Z., 2004. Relationship between injection duration, transporter occupancy and reinforcing strength of cocaine. Eur. J. Pharmacol. 486, 251–257.