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Vol. 303, Issue 1, 211-217, October 2002
Department of Psychiatry, University of Mississippi Medical Center, Jackson, Mississippi (W.L.W., R.R., Z.W., G.A.O., I.A.P.); and Departments of Neurology and Pharmacology, Georgetown University Medical Center, Washington, DC (P.P., A.K.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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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
4
-(4-chlorophenyl)-1-methylpiperidine-3-
-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.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Drugs
that bind dopamine transporters (DATs) and block DA uptake often
function as positive reinforcers (Ritz et al., 1987
; Bergman et al.,
1989). Cocaine is an excellent example of such a compound. There are,
however, a number of compounds that block DA uptake but seem to differ
from cocaine along the dimension of maximum reinforcing effect, or
strength as a positive reinforcer. GBR 12909, for example, is a
highly selective DA uptake blocker (Lewis et al., 1999) that has some
cocaine-like behavioral effects (Spealman et al., 1989; Howell and
Byrd, 1991). Like cocaine, GBR 12909 can serve as a positive reinforcer
to maintain i.v. self-administration by animals (Bergman et al., 1989;
Howell and Byrd, 1991; Skjoldager et al., 1993). However, GBR 12909 seems to be a weaker positive reinforcer than cocaine (Tella et al., 1996; Stafford et al., 2001; Woolverton et al., 2001). Similarly, several local anesthetics, as well as analogs of benztropine, are
effective DA uptake blockers but weaker positive reinforcers than
cocaine (Wilcox et al., 2000; Woolverton et al., 2001).
Research with compounds that block monoamine uptake but vary in
reinforcing strength can help elucidate pharmacological mechanisms that
contribute to their reinforcing strength and enhance our understanding
of the pharmacology of drug abuse. As for any pharmacological effect,
pharmacodynamics and pharmacokinetics are determinants of reinforcing
strength of monoamine transporter ligands. It may be that a compound's
particular combination of monoamine actions influences reinforcing
strength (Roberts et al., 1999). Cocaine is approximately equipotent at
all three monoamine transporters (Carroll et al., 1995), whereas GBR
12909, in contrast, is more selective for the DAT (Lewis et al., 1999).
Alternatively, pharmacological actions involving other nonmonoamine
neurotransmitter systems may act to limit the self-administration of
some DAT ligands (Wilcox et al., 2000; Ranaldi and Woolverton,
2002). Recently, it has been proposed that maximum DAT occupancy
may be an important determinant of the subjective (Volkow et al., 1996,
1997) and reinforcing (Wilcox et al., 2002) effects of cocaine and
other DAT ligands. With regard to pharmacokinetics, GBR 12909 may be a
weaker positive reinforcer than cocaine because of a slower onset of
DAT binding (Pogun et al., 1991). The precise relationship between
these pharmacological factors and reinforcing strength of monoamine
transporter ligands remains largely speculative.
In addition to basic information about brain mechanisms and
reinforcement, compounds that act primarily at monoamine transporters are being widely studied because of their potential as treatment medications for cocaine abuse. Compounds that bind the DAT but are
weaker reinforcers than cocaine may prove useful in this regard (Mello
and Negus, 1996; Howell and Wilcox, 2001). Numerous chemical approaches
have been proposed for the development of novel medications (Newman,
1998; Carroll et al., 1999). Kozikowski and colleagues have synthesized
piperidine-based cocaine analogs that vary in their potencies at the
monoamine transporters (Kozikowski et al., 1998; Petukhov et al.,
2002). One of these compounds, (+)-methyl 4-(4-chlorophenyl)-1-methylpiperidine-3--carboxylate [(+)-CPCA; Fig. 1], has shown promise as a
potential treatment medication. In that study, (+)-CPCA was essentially
equipotent to cocaine in blocking DA and norepinephrine uptake
in vitro in rat brain tissue. However, it was substantially less potent
than cocaine in blocking 5-HT uptake. The compound functioned as a
positive reinforcer in monkeys responding under a fixed ratio schedule of reinforcement, but its relative strength as a positive reinforcer is
unknown. In addition, there is no information currently available concerning the kinetics of transporter occupancy by (+)-CPCA.
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The present study was designed to compare the strength of (+)-CPCA and
cocaine as positive reinforcers in monkeys responding under a
progressive ratio schedule of i.v. self-administration. Progressive
ratio schedules allow the rank ordering of drugs according to their
strength as positive reinforcers (Richardson and Roberts, 1996;
Stafford et al., 1998). Because differences in monoamine transporter
activity have been associated with differences in reinforcing strength
(Roberts et al., 1999), we assessed affinity of (+)-CPCA for monoamine
transporter sites in vitro in monkey brain tissue. Monkey brain tissue
was used to allow more direct comparison to self-administration
results. In addition, because the extent and rate of DAT occupancy may
contribute to reinforcing strength, ex vivo binding methods were used
in rats to compare rate and extent of DAT binding by cocaine and
(+)-CPCA. Ex vivo binding has been used to characterize binding of a
number of radioligands (Stockmeier et al., 1993) and has been applied
to the study of various compounds to the DAT (Scheffel et al., 1991;
Gatley et al., 1999).
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Self-Administration
Subjects and Apparatus.
The subjects were five male
(7.8-11.1 kg) rhesus monkeys (Macaca mulatta). Monkey AV88
was experimentally naïve, whereas the other subjects had
histories of drug self-administration. Most recently, monkey RIk2 had a
history of self-administration of D2 agonists under a progressive ratio
schedule of reinforcement (Woolverton and Ranaldi, 2002).
Monkeys L638 and RJu2 had histories of self-administration of
methamphetamine under a progressive ratio schedule of reinforcement and
pretreatment with a 3-phenyltropane analog (Ranaldi et al., 2000).
Monkey AP78 had a history of self-administration of cocaine/scopolamine
mixtures under a progressive ratio schedule of reinforcement (Ranaldi
and Woolverton, 2002). All monkeys were provided with sufficient food
to maintain stable body weight (150-200 g/day, Teklad 25% Monkey
Diet; Harlan/Teklad, Madison, WI). Water was continuously available. A
vitamin supplement was provided three times per week.
Procedure. Using strict aseptic techniques performed under ketamine and isoflurane anesthesia, a silicone catheter (0.26-cm o.d. × 0.076-cm i.d.; Cole-Parmer Instrument) was implanted into a jugular (internal or external) or femoral vein. Brachial veins were implanted with a tapered microrenethane catheter (0.08-cm o.d. × 0.04-cm i.d.; Braintree Scientific, Braintree, MA). The proximal end was inserted into the vein and threaded to terminate in the vena cava near the right atrium. The distal end of the catheter was passed subcutaneously to exit the monkey between the scapulae. After surgery the catheter was threaded through the spring arm, out the rear of the cubicle and connected to the peristaltic pump. In the event of catheter failure, surgery was repeated using another vein, after the veterinarian confirmed the health of the monkey.
Experimental sessions began at noon each day and were conducted 7 days/week. At the beginning of a session, the white lights were illuminated above both levers. Responding on the right lever under a progressive ratio schedule of reinforcement resulted in the delivery of an injection. Responding on the left lever was counted but had no other programmed consequence. The progressive ratio schedule has been described in detail previously (Wilcox et al., 2000). It consisted of
five components, each made up of four trials, for a total of 20 available trials per day. The response requirement for the first
component was 100 and doubled for each successive component. The same
response requirement was in effect for each trial in a component, and a
trial ended with a 10-s drug injection or the expiration of a 30-min
limited hold (LH). During the injection the lights above both levers
turned from white to red. There was a 30-min time-out (TO) after each
drug injection or the expiration of an LH. If the response requirement
was not completed for two consecutive trials (i.e., the LH expired), or the animal took all 20 injections, the session ended.
In baseline sessions, cocaine (0.1 mg/kg/injection for RJu2, L637, and
RIk2; 0.3 mg/kg/injection for AP13 and AV88) or saline was available
for injection on alternate days until responding was stable (mean ± 2 injections) for at least three consecutive cocaine and saline
sessions. At this point, the session sequence was changed to a double
alternation, i.e., two consecutive cocaine sessions were followed by
two consecutive saline sessions. When responding was again stable, test
sessions were added to the daily sequence between two saline and two
cocaine sessions. To prevent monkeys from learning this sequence and
anticipating sessions, a randomly determined saline or cocaine baseline
session was inserted after every other test session. During test
sessions the monkeys had access to one of various doses of cocaine
(0.01-1.0 mg/kg/injection) or (+)-CPCA (0.01-3.0 mg/kg/injection).
After a test session, a monkey was returned to baseline conditions
until cocaine- and saline-maintained responding was again stable. Doses
of cocaine were tested twice, once the day after a cocaine baseline
session and once the day after a saline baseline session. Because of
limited drug supplies, doses of (+)-CPCA were generally tested once in each monkey. For cocaine, doses were tested in random order with the
first dose for an individual monkey counterbalanced across monkeys. For
(+)-CPCA, doses were tested in an ascending order in the first monkey
tested then in a random order with the first dose for an individual
monkey counterbalanced in the other monkeys.
After testing all doses of (+)-CPCA, the possibility that drug
accumulation over a session decreased progressive ratio responding was
examined in four monkeys by extending the TO after injections to 60 min. Cocaine was tested again, as described above, either at the dose
that maintained the maximum number of injections (L637 and RIk2) as
determined under the 30-min TO, or one dose lower (AP13 and RJu2).
(+)-CPCA was tested again at the dose that maintained maximum
responding in all monkeys. It was expected that if drug accumulation
were limiting maximum progressive ratio responding then lengthening the
TO would increase responding. The fifth monkey (AV88) was not tested in
this condition because of a limited drug supply.
Data Analysis.
Data were analyzed as injections per session
(Wilcox et al., 2000). Means and S.E.M. values were calculated for each
dose of each drug. ED50 values were individually
calculated for cocaine and (+)-CPCA using the linear portion of the
dose-response function (Prism 3.0; GraphPad Software, San Diego, CA).
The maximum number of injections maintained by any dose served as 100%
for this analysis. The actual maximum number of injections, regardless
of dose, was also averaged across all monkeys for both drugs. Mean
ED50 values and S.E.M. values were calculated for
each drug for these measures. Statistical significance of differences
between means was assessed using a paired t test with
significance set at the 0.05 level.
In Vitro Binding
Subjects and Apparatus.
Frozen brain tissue for four rhesus
monkeys was used for the in vitro studies, as described previously
(Woolverton et al., 2000). Each monkey had a history of cocaine
self-administration and had been drug-free for at least 2 months before
sacrifice. During the drug-free period they were maintained under
standard conditions in stainless steel cages with water continuously
available. They were fed a sufficient amount of Teklad Monkey Diet
(Harlan, Indianapolis, IN) to maintain stable body weight, received
fresh fruit 5 days/week, and a chewable multiple vitamin tablet 3 days/week.
Procedure.
No monkeys were sacrificed specifically for this
experiment but had been euthanized previously after all accessible
veins had been used in self-administration studies. For euthanasia, monkeys were sedated with ketamine then given an overdose of i.v. pentobarbital. Brains were collected immediately (within 10 min) after
sacrifice and the caudate nucleus, putamen, frontal cortex, and
cerebellum were dissected (20-30 min) according to the atlas of Snider
and Lee (1961). Immediately after dissection, tissue was frozen on
aluminum foil over solid CO2 for 30 min, with no additional preparation, and then placed into a 
80°C freezer until assay.
) and centrifuged at 20,000g for 20 min at 4°C. The resulting pellet was resuspended in fresh buffer and
centrifuged an additional one or two times, depending on the assay.
After centrifugation, the pellet was suspended at the appropriate
tissue concentration for displacement or saturation assays.
Specific conditions for displacement and saturation assays were as
previously published with minor changes (Table
1; Woolverton et al., 2000). For
displacement studies the tissue was added to assays containing the
radioligand and various concentrations of cocaine or (+)-CPCA dissolved
in assay buffer. For saturation studies, the tissue was incubated with
varying concentrations of [3H]CFT (0.1-25.6
nM), [3H]nisoxetine (0.1-12.8 nM), and
[3H]paroxetine (0.006-25.6 nM). For
displacement studies, concentrations of cocaine and (+)-CPCA ranged
between 0.1 nM and 1 mM. For both saturation and displacement studies
all assay tubes were brought to their final volume (1.0 ml for
[3H]CFT,
[3H]paroxetine; 500 µl for
[3H]nisoxetine with the addition of buffer).
All assays were initiated with the addition of tissue. Assays were
incubated under conditions described in Woolverton et al. (2000).
Reactions were terminated by rapid vacuum filtration using a 24-well
cell harvester (Brandel Inc., Gaithersburg, MD) through presoaked
GF/C filters (Whatman, Maidstone, UK; Table 1). The filters were rinsed
with ice-cold buffer and deposited into Top Count deep-well plates
(Packard Instrument Company, Inc., Downers Grove, IL). Five hundred
microliters of Microscint-20 cocktail (Packard Instrument Company,
Inc.) was added to each well. Bound radioactivity was determined using
a Top Count scintillation counter (Packard Instrument Company, Inc.). Protein levels in tissue homogenate samples were determined using the
bicinchoninic acid method (Smith et al., 1985; kits from Pierce Chemical, Rockford, IL). Absorbance (at 560 nm) was measured on a
spectrophotometer (Beckman Coulter, Inc., Fullerton, CA). All saturation and displacement assays were performed in triplicate.
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Data Analysis.
Radioligand binding data were initially
reduced and analyzed using iterative curve fitting (Prism 3.0; GraphPad
Software). Kd and
Bmax values, and their 95% confidence
intervals (CIs), were derived from saturation studies. To compare the
goodness-of-fit of one-site models to two-site models, one-site and
two-site models with all Hill coefficients fixed to 1 were fit to
displacement data. The one-site model was assumed unless the mean
square error was significantly reduced by using a two-site model
(p < 0.05 using a univariate F test).
Ki values and their 95% CIs were
calculated for each compound from displacement studies.
Ex Vivo Binding
DAT binding was studied ex vivo using methods similar to those
previously published using mice (Scheffel et al., 1991; Stathis et al.,
1995; Gatley et al., 1999).
Subjects and Apparatus. The subjects were male Sprague-Dawley rats weighing between 250 and 300 g. They were initially housed in groups of three in plastic cages and with a 12-h light/dark cycle (lights on at 6:00 AM). Food and water were available ad libitum.
Procedure. Drugs were given i.v. via a surgically implanted catheter. For surgery, rats were anesthetized with pentobarbital (50 mg/kg i.p.) and a femoral catheter was implanted using standard techniques. The exteriorized tip of the catheter was sealed by heating. After surgery, they were housed individually for 48 h then used experimentally.
To establish relative potency for DAT occupancy, injections of dose of cocaine or (+)-CPCA were given before [3H]CFT to permit maximum (or equilibrium) binding of cocaine and (+)-CPCA to the DAT before the injection of [3H]CFT. Initially, catheterized rats were placed in a plastic restrainer and injected i.v. (0.4 ml/rat/10 s) with either 10 µmol/kg cocaine or 30 µmol/kg (+)-CPCA. These doses were selected based upon preliminary studies with cocaine and the in vitro potency ratio between cocaine and (+)-CPCA. At various time points after drug injection (0.5-30 min), rats were injected with [3H]CFT, also i.v. over 10 s. Because preliminary studies showed that the striatal/cerebellar ratio (S/C) of [3H]CFT reached a maximum 45 min after injection of [3H]CFT, rats were decapitated at this time point. After decapitation, brains were removed and dissected into striatum (high DAT density) and cerebellum (no DAT, nonspecific binding). Striatum and cerebellum were weighed and placed into separate 5-ml glass vials. Solvable (10 µl/mg tissue) was added and the vial was allowed to sit for 24 h at room temperature. After 24 h, glacial acetic acid (1 µl/mg tissue) was added and 100 µl of the tissue solution was immediately pipetted into each well of 24-well scintillation plates (3-4 wells/sample). Microscint-20 cocktail (500 µl) was then added to each well and the plate was sealed. This preparation was allowed to sit for 4 h to further solubilize tissue and reduce chemiluminescence of the Microscint-20 cocktail. Radioactivity was then counted. Complete dose-response functions were then determined for each drug using the time points at which the decrease in [3H]CFT was maximal. ED50 values were calculated for reduction in [3H]CFT binding. To establish the time course of DAT binding, an injection of a selected dose of cocaine or (+)-CPCA was given at the time point at which [3H]CFT binding was asymptotic. The decrease in binding was measured at various time points after drug injection and compared with the same points after saline injection (Stathis et al., 1995). Specifically, saline or ED50 doses of
cocaine or (+)-CPCA were given 45 min after injection of
[3H]CFT. Animals were decapitated at various
time points (0.5-120 min) after injection of test drugs.
Data Analysis.
For each ligand, S/C was calculated. Data
were normalized to S/C 1 so that complete inhibition of binding
approached zero. Transporter occupancy was calculated using the
equation % occupancy = (A x/A B) × 100 (Gatley et al., 1999). In this equation, A and
x are S/C measured after injection of radioligand alone and
drug plus radioligand, respectively. B is the S/C measured after a high dose of cold GBR 12909, a selective DAT ligand, which is
assumed to reflect 100% occupancy of the transporter. The difference between B and 1.0 presumably reflects differences in
nonspecific binding. ED50 values (the dose of
competing drug displacing half the specific binding) and maximum
occupancy were calculated using iterative curve fitting (Prism 3.0;
GraphPad Software). Statistical significance of
ED50 differences was assessed using Student's t test with significance set at the p < 0.05 level.
Drugs.
Cocaine HCl was provided by the National Institute on
Drug Abuse (Rockville, MD) and was dissolved in 0.9% saline for
self-administration. (+)-CPCA was synthesized as described by
Kozikowski et al. (1998) and was also dissolved in saline. Doses are
expressed as the salt forms of the drugs. Molecular masses are
339 µg/µmol for cocaine and 304 µg/µmol for (+)-CPCA.
Radioligands were purchased from PerkinElmer Life Sciences (Boston,
MA). For binding studies, drugs were mixed fresh before each assay.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Self-Administration. In baseline sessions, cocaine maintained an average of between 8.2 (AP13) and 17.8 (RIk2) injections/session, whereas saline maintained between 1.0 (AP13) and 2.0 (L637) injections/session. Over the course of the experiment, responding in baseline sessions did not change systematically across monkeys (data not shown).
Cocaine functioned as a reinforcer in all monkeys and the mean dose-response function was asymptotic (Fig. 2). On an individual basis, responding increased with dose to an asymptote in four of the five monkeys and decreased again in the fifth (L637) at 1.0 mg/kg/injection (data not shown). (+)-CPCA also functioned as a positive reinforcer in all monkeys, and both mean and individual dose-response functions were biphasic. The mean ED50 was 0.06 mg/kg/inj (0.01 S.E.M.) for cocaine and 0.21 mg/kg/inj (0.08 S.E.M.) for (+)-CPCA. On a molar basis, ED50 values were 0.17 µmol/kg/inj (0.04 S.E.M.) for cocaine and 0.68 µmol/kg/inj (0.25 S.E.M.) for (+)-CPCA. The difference was not statistically significant (p = 0.08), primarily because ED50 values for the two drugs were comparable in one monkey (L637). The mean maximum number of injections per session maintained was 16.5 (1.69 S.E.M.) for cocaine and 12.1 (1.48 S.E.M.) for (+)-CPCA. Differences between drugs in injection maximums were statistically significant (p = 0.04). When the TO after injection was increased to 60 min the maximum was either unaffected or decreased (Table 2).
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In Vitro Binding.
Results of saturation studies using the
different 3H-ligands in rhesus monkey brain are
presented in Table 3. Both compounds inhibited binding of all three radioligands in a concentration-related manner. Displacement was consistent with a one-site model for all three
radioligands. Cocaine had about a 2-fold higher affinity for the
[3H]CFT site than did (+)-CPCA, whereas the
compounds had similar affinities for the
[3H]nisoxetine site (Table
4). For displacement of
[3H]paroxetine, the affinity of cocaine was
approximately 15-fold higher than that of (+)-CPCA.
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Ex Vivo Binding.
The maximum decrease in
[3H]CFT binding by cocaine was seen when
cocaine was given 3 min before [3H]CFT (data
not shown). For (+)-CPCA, this value was 10 min. When various doses
were administered at these pretreatment times, both cocaine and
(+)-CPCA inhibited the binding of [3H]CFT in a
dose-related manner. Occupancy of the DAT increased in a dose-related
manner (Fig. 3). The
ED50 for cocaine was 8.82 µmol/kg and for CPCA
was 34.3 µmol/kg. This difference was statistically significant
(p = 0.03). Maximum occupancy was comparable for both drugs (p = 0.48). When the ED50
of each drug was given at various times before sacrifice, DAT occupancy
was greater for cocaine than for (+)-CPCA 2 min after injection (Fig.
4). The effects of the two drugs were not
different at other time points.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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As in previous studies using this and other progressive ratio
schedules (Stafford et al., 1998; Wilcox et al., 2000), cocaine functioned as a positive reinforcer and responding increased with dose
to an asymptote. (+)-CPCA also functioned as a positive reinforcer under the present progressive ratio schedule, confirming and extending previous self-administration results with this compound in monkeys maintained under a fixed ratio schedule (A. Kozikowski, personal communication). Responding maintained by (+)-CPCA increased with dose
then decreased at the highest dose tested. Although only tested in one
monkey, it is likely that cocaine-maintained responding would have
decreased again had doses of 1.0 mg/kg/injection or higher been tested.
In all but one monkey, cocaine was approximately 4-fold more potent
than (+)-CPCA as a reinforcer. Maximum levels of responding were higher
for cocaine than for (+)-CPCA. Doubling the TO after an injection from
30 to 60 min failed to increase cocaine or (+)-CPCA-maintained
responding, arguing that the maximum response maintained was not
limited by the duration of action for either drug. These findings
support the conclusion that cocaine was a stronger positive reinforcer
than (+)-CPCA.
Clearly, reinforcing strength can vary with behavioral conditions
(Katz, 1990). Because behavioral conditions were constant across the
two drugs tested in the present experiment, it is possible to examine
pharmacological mechanisms that may have contributed to differences in
reinforcing strength. Pharmacodynamically, it has been suggested that
the mix of effects on monoamine neurotransmission may influence
reinforcing strength. More specifically, increased 5-HT relative to DA
activity has been associated with diminished reinforcing strength
(Vanover et al., 1992; Roberts et al., 1999). In monoamine uptake
studies conducted in vitro in rat brain tissue, (+)-CPCA has been
reported to have DA and norepinephrine activity comparable with that of
cocaine, but to have diminished 5-HT activity (A. Kozikowski, personal
communication). In vitro binding data in the present study extend that
observation to transporter binding in monkey brain tissue. Other
monoamine actions being equal, then, one would expect diminished 5-HT
activity to enhance, or at least not diminish, the reinforcing strength
of (+)-CPCA. That is, these findings argue against a conclusion that
the mix of monoamine actions of (+)-CPCA was responsible for its
reduced reinforcing strength relative to cocaine. It should also be
considered that (+)-CPCA may have actions that cocaine lacks on other
neurotransmitter systems that influence self-administration. For
example, it has been suggested that sodium channel actions may limit
the reinforcing effects of local anesthetics (Wilcox et al., 2000).
Additionally, the reduced reinforcing strength of benztropine analogs
that are DAT ligands may be related to anticholinergic actions (Ranaldi and Woolverton, 2002). Although its actions on other brain
neurotransmitter systems could influence the self-administration of
(+)-CPCA, it has little or no affinity for other receptor binding sites
(A. Kozikowski, personal communication).
Occupancy of the DAT has also been proposed to play a role in
subjective effects and reinforcing effects of DAT ligands. Volkow et
al. (1997) reported that the subjective effects of cocaine increased
with DAT occupancy and that doses commonly abused by humans occupied 60 to 77% of the DAT sites. Wilcox et al. (2002) reported DAT occupancies
of 64 to 75% and 94 to 99% for doses of cocaine or RTI-113,
respectively, that maintained maximum rates of responding in monkeys.
In the present study, the potency relationship between cocaine and
(+)-CPCA was comparable in the self-administration and the occupancy
assays. In addition, DAT occupancy by cocaine was about 60% at a dose
of 10 µmol/kg (3.4 mg/kg), well within the range of doses shown to
maintain responding in rats under a progressive ratio schedule of
reinforcement (Roberts et al., 1999). Based on these data, there seems
to be a good correspondence in the relationship potency in occupying
the DAT and potency as a reinforcer that holds across species. However,
in the present study cocaine and (+)-CPCA differed in their strength as
reinforcers but exhibited comparable maximum occupancies of the DAT.
Thus, the hypothesis that relative reinforcing strength was directly related to maximum occupancy of the DAT was not supported. Data from
previous studies do not address this hypothesis. Additional research is
required to further examine relationship between reinforcing strength
and maximum DAT occupancy. Given that reinforcing strength is only one
component of abuse liability, any relationship between DAT occupancy
and abuse liability remains to be established.
Onset of action has also been proposed to be an important
determinant of the reinforcing effect of drugs. Perhaps the strongest data in support of this notion have been collected in humans. More
rapid onset of action has been associated with increased subjective
effects of drugs in human subjects (deWit et al., 1993; Marsch et al.,
2001). Although there is the suggestion of this effect in the animal
literature (Balster and Schuster, 1973), a systematic analysis of the
relationship between onset of action and reinforcing strength has not
been undertaken. In a preliminary study, we found that monkeys given a
choice between 0.1 mg/kg cocaine injected over 10 s and this same
dose of cocaine injected over 30 or 100 s, preferred the more
rapid injection. In the present study, cocaine occupied a higher
proportion of DATs than did (+)-CPCA at 2 min after injection. At other
time points, DAT occupancy was comparable for the two drugs. The
precise mechanism accounting for this difference, e.g., differences in
distribution or binding at the site of action, are unclear at this
point. Assuming that the kinetic difference observed at the
ED50 is comparable across the dose-response
function, these data support the hypothesis that the difference in
reinforcing strength between cocaine and (+)-CPCA is related to the
slower onset of DAT occupancy by (+)-CPCA. More generally, these data
suggest that reinforcing strength is directly related to rate of DAT
occupancy over a time frame that is as short as the first 3 min after
injection. Furthermore, our findings suggest that between-drug
differences in rate of DAT occupancy as small as 1 min can influence
relative reinforcing strength. Obviously, additional research is
required to support or refute this conclusion. Nevertheless, the
present data begin to provide an empirical time frame for the
hypothesis that rate of onset contributes to reinforcing strength.
It has been amply demonstrated that among DAT ligands, potency as
a positive reinforcer is correlated with DAT affinity (Ritz et al.,
1987; Bergman et al., 1989). However, the amount of behavior maintained
by a drug is more nearly predicted by its relative strength as a
reinforcer than by its potency. Clearly, strength as a reinforcer is
multifaceted and determined by many pharmacological factors, only one
of which is potency. The present experiment begins to quantify some of
the factors, most particularly kinetics of onset, which may contribute
to the reinforcing strength of monoamine transporter ligands. In
addition to these basic research issues, the observation that (+)-CPCA
can function a positive reinforcer when self-administered i.v. by
monkeys raises the possibility of abuse liability in humans. This could
be a consideration in its development as a treatment medication for
cocaine abuse. However, the observation that (+)-CPCA is a weaker
positive reinforcer than cocaine under a progressive ratio schedule
suggests lower abuse liability than cocaine. This conclusion must be
drawn with the caveat that this relative strength relationship may vary
with behavioral conditions. It has been proposed that a desirable
agonist pharmacotherapy for cocaine abuse should have some reinforcing effect, to encourage patient compliance, and a slow onset of action (Gorelick, 1997). The present data support further examination of this
compound in the pharmacotherapeutic context.
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Acknowledgments |
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We thank Kenneth Pace and Chaleeta Arender for technical assistance.
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Footnotes |
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Accepted for publication May 29, 2002.
Received for publication April 24, 2002.
This research was supported by National Institute on Drub Abuse Grants DA10352, DA00161 (to W.L.W.), MHAG02031 (to G.A.O.), and DA11548 (to A.K.). The data were presented at the 2002 meeting of the American Society of Pharmacology and Experimental Therapeutics, New Orleans, LA.
DOI: 10.1124/jpet.102.037812
Address correspondence to: Dr. William L. Woolverton, Department of Psychiatry, University of Mississippi Medical Center, 2500 N. State St., Jackson, MS 39216-4505. E-mail: wwoolverton{at}psychiatry.umsmed.edu
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Abbreviations |
|---|
DAT, dopamine transporter;
DA, dopamine;
(+)-CPCA, (+)-methyl
4-(4-chlorophenyl)-1-methylpiperidine-3--carboxylate;
LH, limited
hold;
TO, time-out;
5-HT, 5-hydroxytryptamine;
CFT, 2-carbomethoxy-3-(4-fluorophenyl)tropane);
CI, confidence
interval;
S/C, striatal/cerebellar ratio;
inj, injection;
GBR 12909, 1-[2-[bis(4-fluorophenyl)methoxy]ethy]-4-(3-phenylpropyl)piperazine.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-(4'-substituted phenyl)tropane-2-carboxylic acid ester and amide analogues. New high-affinity and selective compounds for the dopamine transporter.
J Med Chem
38:
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