Development of a rationally designed, low abuse potential, biogenic amine releaser that suppresses cocaine self-administration

Article abstract of DOI:10.1124/jpet.104.082503

Convergent lines of evidence support a dual deficit model of stimulant withdrawal, where reductions in synaptic dopamine (DA) and 5-hydroxytryptamine (serotonin) (5-HT) contribute to dysphoria, drug craving, and relapse. Thus, we predicted that a nonamphetamine compound with substrate activity at DA and 5-HT transporters (i.e., a dual DA/5-HT releaser) would be an effective medication for treating stimulant addictions. Ideally, this type of medication would alleviate withdrawal symptoms, suppress cocaine self-administration, and lack side effects commonly associated with central nervous system stimulants. In the present work, more than 350 compounds were screened in vitro for activity as substrate-type releasing agents at DA, 5-HT, and norepinephrine transporters. These efforts identified PAL-287 (1-napthyl-2-aminopropane) as a nonamphetamine compound with potent substrate activity at biogenic amine transporters. In vivo microdialysis in rats demonstrated that PAL-287 (1-3 mg/kg i.v.) increased extracellular DA and 5-HT in frontal cortex, but effects on 5-HT were somewhat greater. PAL-287 induced substantially less locomotor stimulation than (+)-amphetamine, a drug that increases only extracellular DA. Administration of high-dose (+)-methamphetamine or (±)-3,4-methylenedioxymethamphetamine to rats produced long-lasting depletion of cortical 5-HT, whereas PAL-287 (18 mg/kg i.p. × 3) did not. PAL-287 displayed little or no reinforcing properties in rhesus monkeys trained to self-administer cocaine, yet PAL-287 produced a dose-dependent decrease in responding for cocaine when infused at a dose of 1.0 mg/kg/h. Collectively, the findings reported here demonstrate that nonamphetamine monoamine releasing agents such as PAL-287 might be promising candidate medications for the treatment of stimulant dependence.

Full text of DOI:10.1124/jpet.104.082503

0
022-3565/05/3133-1361–1369
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
U.S. Government work not protected by U.S. copyright
JPET 313:1361–1369, 2005
Vol. 313, No. 3
82503/1199560
Printed in U.S.A.
Development of a Rationally Designed, Low Abuse Potential,
Biogenic Amine Releaser That Suppresses Cocaine Self-
Administration
Richard B. Rothman, Bruce E. Blough, William L. Woolverton, Karen G. Anderson,
S. Stevens Negus, Nancy K. Mello, Bryan L. Roth, and Michael H. Baumann
Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of
Health, Department of Health and Human Services, Baltimore, Maryland (R.B.R., M.H.B.); Chemistry and Life Sciences Group,
Research Triangle Institute International, Research Triangle Park, North Carolina (B.E.B.); Department of Psychiatry and
Pharmacology, University of Mississippi Medical Center, Jackson, Mississippi (W.L.W., K.G.A.); Alcohol and Drug Abuse
Research Center, Harvard Medical School, McLean Hospital, Belmont, Massachusetts (S.S.N., N.K.M.); and Departments of
Biochemistry, Neurosciences, and Psychiatry, and the National Institutes of Mental Health Psychoactive Drug Screening
Program, Case Western Reserve University School of Medicine, Cleveland, Ohio (B.L.R.)
Received December 20, 2004; accepted February 7, 2005
ABSTRACT
Convergent lines of evidence support a dual deficit model of in rats demonstrated that PAL-287 (1–3 mg/kg i.v.) increased
stimulant withdrawal, where reductions in synaptic dopamine (DA) extracellular DA and 5-HT in frontal cortex, but effects on 5-HT
and 5-hydroxytryptamine (serotonin) (5-HT) contribute to dyspho- were somewhat greater. PAL-287 induced substantially less loco-
ria, drug craving, and relapse. Thus, we predicted that a nonam- motor stimulation than (ϩ)-amphetamine, a drug that increases
phetamine compound with substrate activity at DA and 5-HT only extracellular DA. Administration of high-dose (ϩ)-metham-
transporters (i.e., a dual DA/5-HT releaser) would be an effective phetamine or (Ϯ)-3,4-methylenedioxymethamphetamine to rats
medication for treating stimulant addictions. Ideally, this type of produced long-lasting depletion of cortical 5-HT, whereas PAL-
medication would alleviate withdrawal symptoms, suppress co- 287 (18 mg/kg i.p. ϫ 3) did not. PAL-287 displayed little or no
caine self-administration, and lack side effects commonly associ- reinforcing properties in rhesus monkeys trained to self-administer
ated with central nervous system stimulants. In the present work, cocaine, yet PAL-287 produced a dose-dependent decrease in
more than 350 compounds were screened in vitro for activity as responding for cocaine when infused at a dose of 1.0 mg/kg/h.
substrate-type releasing agents at DA, 5-HT, and norepinephrine Collectively, the findings reported here demonstrate that nonam-
transporters. These efforts identified PAL-287 (1-napthyl-2-amin- phetamine monoamine releasing agents such as PAL-287 might
opropane) as a nonamphetamine compound with potent sub- be promising candidate medications for the treatment of stimulant
strate activity at biogenic amine transporters. In vivo microdialysis dependence.
The abuse of illicit stimulants such as cocaine and meth- United States (Banken, 2004). Importantly, the abuse of
amphetamine remains a major public health problem in the central nervous system stimulants negatively impacts public
health, through the spread of human immunodeficiency vi-
rus-1, hepatitis B and C, and drug-resistant tuberculosis
Kresina et al., 2004). Despite the extent of the stimulant
abuse crisis, development of medications to treat stimulant
dependence has not been successful (Shearer and Gowing,
This research was supported in part by P01-DA14528 (to N.K.M.), R01-
DA02519 (to N.K.M.), and K05-DA00101 (to N.K.M.) from the National Institute
on Drug Abuse, National Institutes of Health. The self-administration study was
supported by National Institute on Drug Abuse Grant R01 DA-10352 (to W.L.W.).
W.L.W. is the recipient of National Institute on Drug Abuse Senior Scientist
Award K05-DA15343. B.E.B. was supported by National Institute on Drug Abuse
Grant R01 DA12970. R.B.R. and M.H.B. were supported by the Intramural
Research Program, National Institute on Drug Abuse.
(
2004).
The lack of effective medications for stimulant dependence
renders discussions of an “ideal” pharmacotherapeutic for
stimulant addiction somewhat hypothetical. Nevertheless, a
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.104.082503.
ABBREVIATIONS: DA, dopamine; 5-HT, 5-hydroxytryptamine (serotonin); PAL-287, 1-napthyl-2-aminopropane; NE, norepinephrine; RTI-229,
␤-(4-iodophenyl)tropane-2␤-pyrrolidine carboxamide; GBR 12909, 1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine;
3
HPLC, high-performance liquid chromatography; ECD, electrochemical detection; (Ϯ)-MDMA, (Ϯ)-3,4-methylenedioxymethamphetamine; FR,
fixed ratio; VR, variable ratio; ANOVA, analysis of variance; DAT, dopamine transporter; SERT, serotonin transporter; NET, norepinephrine
transporter; PPH, primary pulmonary hypertension; mCPP, m-chlorophenylpiperazine.
1361

1
362
Rothman et al.
reasonable conceptualization distinguishes between medica- reveal that PAL-287 is a monoamine releaser capable of
tions that reduce drug-seeking behavior and medications suppressing cocaine self-administration, with minimal neu-
that reduce relapse (van den Brink and van Ree, 2003). The rotoxic and reinforcing actions.
former can be readily tested in animal models that measure
drug self-administration, whereas the latter can be tested in
models that are somewhat less validated (Katz and Higgins,
Materials and Methods
Animals
2
003). It seems possible that medications that reduce drug-
seeking behavior might also prevent relapse due to shared
For in vivo microdialysis experiments, male Sprague-Dawley rats,
neurochemical properties between the medication and the purchased from Charles River Laboratories (Wilmington, MA),
weighing 280 to 320 g, were single-housed (lights on from 7:00 AM to
abused stimulant. Agonist substitution therapy illustrates
7
:00 PM) with food and water freely available. For in vitro neuro-
this approach. Often described as neurochemical normaliza-
tion therapy (Rothman et al., 2002a), agonist substitution
therapy has generated effective treatments for nicotine
dependence (Henningfield, 1995) and opioid dependence
transmitter release assays and neurotoxicity studies, rats were
group-housed. Rats were maintained in facilities accredited by the
American Association of the Accreditation of Laboratory Animal
Care, and the procedures described herein were carried out in accor-
dance with the Animal Care and Use Committee of the National
Institute on Drug Abuse Intramural Research Program.
(
Kreek, 1996).
We, and others, have advocated the use of monoamine
releasers such as amphetamine as “agonist” therapies for
All rhesus monkeys (Macaca mulatta) studied at the University of
stimulant dependence (Rothman et al., 2002a; Grabowski et Mississippi Medical Center (Jackson, MS) and at the Alcohol and
al., 2004). Preclinical studies strongly support this approach Drug Abuse Research Center (McLean Hospital, Belmont, MA) were
maintained in accordance with guidelines provided by the National
(
Wojnicki et al., 1999). Negus and Mello (2003a), for example,
Institutes of Health Committee on Laboratory Animal Resources.
Each protocol was reviewed and approved by the Institutional Ani-
mal Care and Use Committee.
demonstrated that (ϩ)-amphetamine infusions decrease co-
caine self-administration behavior in monkeys, with minimal
effects on food-maintained behavior. Perhaps more impor-
tantly, Grabowski et al. (2001) and Shearer et al. (2003)
showed that (ϩ)-amphetamine is an effective treatment ad-
junct for reducing illicit cocaine use in cocaine-dependent
human patients.
Synthesis of PAL-287
2
-Naphthaldehyde (11.0 g; 0.0704 mol) was added to a stirred
mixture of ammonium acetate (5.42 g; 0.0704 mol) in nitroethane
101 ml) at 120°C under nitrogen. After refluxing overnight, the
reaction mixture was cooled to room temperature, and excess nitro-
(
A major impediment to the use of amphetamine-type drugs
as treatments is their high abuse potential. A wealth of ethane was removed under reduced pressure. The resulting orange/
information has established that DA release from mesolimbic brown solid was dissolved in a solution of 10% methylene chloride in
nerve terminals mediates the motor stimulant and addictive diethyl ether (30 ml) and washed with brine (2 ϫ 75 ml) and 1.0 M
HCl (3 ϫ 50 ml). The organic layer was dried over anhydrous sodium
sulfate and filtered through celite, and volatiles were removed under
properties of drugs like amphetamine (Wise, 1996). We sug-
gested the possibility of designing novel monoamine releas-
ers with reduced abuse potential; this could be accomplished
by altering the relative potency of these drugs at DA trans-
reduced pressure affording 12.2 g (81%) of crude product, which was
1
pure enough for the next reaction. H NMR (CDCl ) ␦ (ppm): 8.26 (s;
3
1
H, CHCH NO ), 7.91 (m; 4H, ArH), 7.56 (m, 3H, ArH), 2.55 (s, 3H,
3 2
porters versus 5-HT transporters (Rothman and Baumann,
000). The rationale for this idea is based on the knowledge
that 5-HT releasing agents such as fenfluramine lack stim-
CHCCH NO ).
3
2
2
To a stirred solution of 2-(2-nitro-propenyl)-naphthalene (12.2 g;
.0568 mol) and methylene chloride (65 ml) under nitrogen at room
0
ulant properties and are not self-administered (Woods and temperature in a 500-ml round-bottom flask equipped with a reflux
Tessel, 1974). Additionally, fenfluramine reduces locomotor condenser and addition funnel, 1.0 M lithium aluminum hydride
and rewarding actions of DA releasers (Bendotti et al., 1980; (170.5 ml) was added dropwise at a rate to maintain reflux but avoid
overflow. After addition was complete, the reaction was heated to
Baumann et al., 2000; Rothman and Baumann, 2000). Stud-
80°C and refluxed for 48 h. The reaction was quenched dropwise with
ies using coadministered phentermine and fenfluramine
demonstrate that agents that increase both extracellular DA
and 5-HT in rat central nervous system produce minimal
motor activity (Baumann et al., 2000) and are not rewarding
deionized water and combined with a solution of Rochelle’s salt (150
ml) and stirred under nitrogen overnight. The solution was then
filtered through celite, extracted with a 3:1 solution of methylene
chloride/tetrahydrofuran (4 ϫ 100 ml), and dried over anhydrous
sodium sulfate, and volatiles were removed under reduced pressure
affording 11.3 g of crude free base. The free base was combined with
(
Rea et al., 1998) or reinforcing (Glatz et al., 2002). Further-
more, the phentermine/fenfluramine mixture suppresses co-
caine self-administration in rodents and nonhuman primates 1.0 M HCl in diethyl ether (61 ml), forming a precipitate that was
Glowa et al., 1997; Glatz et al., 2002). Consistent with other filtered and recrystallized overnight in the freezer with methanol/
findings (Walsh and Cunningham, 1997), the aforementioned ethyl acetate, affording 8.47g (67%) of pure white crystals. Melting
(
point: 205.2–205.5°C; Anal. Calcd for C13
H
15N⅐HCl: C, 70.42; H, 7.27;
data support the hypothesis that elevations in synaptic 5-HT
can counteract stimulant and reinforcing effects mediated by
elevations in synaptic DA (Czoty et al., 2002; Daw et al.,
1
N, 6.32. Found: C, 70.01; H, 7.39; N, 6.24. H NMR (CD OD) ␦ (ppm):
3
7
1
(
.86 (m; 3H, ArH), 7.73 (s; 1H, ArH), 7.50 (m; 2H, ArH), 7.39 (d; J ϭ
.7 Hz, 1H, ArH), 3.64 (X of ABX; m, 1H, CH CHCH NH ), 3.13, 2.99
2
3
2
2
002; Burmeister et al., 2004).
AB of ABX; JAB ϭ 13.6 Hz, JAX ϭ 6.5 Hz, JBX ϭ 8.1 Hz, 2H,
In the present study, we sought to identify a nonamphet-
CH CHCH NH ), 1.30 (d; J ϭ 6.6 Hz, 3H, CH CHCH NH ).
2
3
2
2
3
2
amine agent that evokes transporter-mediated release of DA
and 5-HT with similar potency in nervous tissue. The result- In Vitro Release Assays
ing compound, PAL-287, was tested using a variety of in vivo
bioassay methods, including intracerebral microdialysis in
3
3
3
In vitro [ H]DA [ H]NE, and [ H]5-HT release assays were per-
formed according to published methods (Rothman et al., 2002b).
rat cortex, motor stimulant activity in rats, and drug self- Briefly, a crude synaptosomal tissue preparation from rat caudate
administration behavior in rhesus monkeys. Our findings (DA release assay), or from whole brain minus caudate (NE and

Releasing Agents Suppress Cocaine Self-Administration
1363
5
-HT release assays) was obtained by homogenization of freshly Drug Self-Administration Studies Carried Out at the
excised tissue in ice-cold 10% sucrose using 12 strokes of a Potter- University of Mississippi Medical Center
Elvehjem homogenizer, followed by centrifugation at 1000g for 10
Subjects and Apparatus. The subjects were four male rhesus
monkeys (Macaca mulatta) weighing between 10 and 10.7 kg at the
beginning of the study. All the monkeys had histories of cocaine
self-administration. Most recently, under the conditions of the
present experiment, monkey AP78 had been tested with atomox-
etine, monkey M341 with pseudoephedrine, and monkey AP01 with
amphetamine analogs. Monkey 12884 was tested with cocaine and
sibutramine under a progressive ratio schedule (our unpublished
data). All monkeys were provided with sufficient food to maintain
stable body weight (120–180 g/day, Teklad 25% Monkey Diet; Har-
lan/Teklad, Madison, WI) and had unlimited access to water. Fresh
fruit and a vitamin supplement were provided daily and three times
a week, respectively.
min. Supernatants were retained on ice and used immediately in
release assays. All assays were performed in Krebs-phosphate
buffer, pH 7.4, which contained 154.4 mM NaCl, 2.9 mM KCl, 1.1
mM CaCl , 0.83 mM MgCl , 5 mM glucose, 1 mg/ml ascorbic acid,
2
2
and 50 ␮M pargyline. Reserpine (1 ␮M) was added to the sucrose
3
solution and assay buffer. [ H]NE release assays were performed in
3
the presence of 5 nM RTI-229 to prevent reuptake of [ H]NE into
3
DAergic nerves. [ H]5-HT release assays were performed in the
presence of 100 nM nomifensine and 100 nM GBR 12909 to prevent
3
reuptake of [ H]5-HT into NEergic and DAergic nerves. For release
assays, synaptosomal preparations were incubated to steady state
3
3
3
with 5 nM [ H]DA (30 min), 7 nM [ H]NE (60 min), or 5 nM [ H]5-HT
60 min). Synaptosomes preloaded with neurotransmitter were
added to test tubes containing test drugs and incubated for 5 min
(
The monkeys were individually housed in the experimental cubi-
3
cles (1.0 m ; PlasLabs, Lansing, MI). Each monkey was fitted with a
3
3
3
(
[ H]DA and [ H]5-HT) or 30 min ([ H]NE). At the designated time,
stainless steel harness attached by a tether to the rear wall of the
cubicle. The front door of the cubicle was made of transparent plastic
and the remaining walls were opaque plastic. Two response levers
the assay was filtered using a PerkinElmer Filtermate harvester.
Nondisplaceable tritium was measured by incubations in the pres-
3
ence of 100 ␮M tyramine for [ H]5-HT release and 10 ␮M tyramine
(
PRL-001; BRS/LVE, Beltsville, MD) were mounted on the inside of
3
3
for [ H]DA and [ H]NE release. Data of three experiments were
pooled and fit to the two-parameter logistic equation of the best-fit
estimates of the EC50 and Emax using MLAB-PC (Civilized Software,
Silver Spring, MD).
the door. Four jeweled stimulus lights, two red and two white, were
mounted above each lever. A peristaltic infusion pump (Cole-Parmer
Instrument Co., Chicago, IL) delivered drug injections. A Macintosh
computer with custom interface and software controlled all events in
an experimental session.
In Vivo Microdialysis Experiments
Procedure. Details of the procedure have been previously pub-
lished (Wee et al., 2004). Briefly, monkeys were implanted with i.v.
silastic catheters and allowed to self-administer drugs by pressing
the right lever (25 lever presses/injection; FR25) for 2 h/day, begin-
ning at noon, 7 days/week. At the start of a session, the white lights
were illuminated above both levers, and responding on the right
lever resulted in the delivery of a drug injection for 10 s. During the
injection, the white lights were extinguished and the red lights were
illuminated. Pressing the left lever was counted but had no other
programmed consequence.
In baseline sessions, cocaine (0.03 mg/kg/injection for AP01, AP78,
and M341; 0.01 mg/kg/injection for L638) or saline was made avail-
able. Test sessions were arranged among baseline sessions in a way
that a test session came after two different baseline sessions, that is,
C-S-T or S-C-T where C, S, and T denote cocaine, saline, and test
sessions, respectively. A test session was identical to a baseline
session, except that saline or one of various doses of cocaine (0.001–
Surgical implantation of indwelling jugular catheters and intra-
cerebral guide cannulae was carried out as described previously
(
Baumann et al., 2000). Guide cannulae were aimed at the prefrontal
cortex according to coordinates ML, 2.5 mm and AP, ϩ3.0 mm
relative to bregma, DV, 0.8 mm relative to dura. Rats were allowed
1
week to recover from surgery. On the evening before testing,
extension tubes were connected to catheters, and microdialysis
probes (3 ϫ 0.5-mm exchange surface, CMA/12; CMA/Microdialysis,
Solna, Sweden) were inserted into the frontal cortex via guide can3-
nulae. Each rat was attached to a tether and placed into a 40-cm
Plexiglas arena equipped with photobeams that allowed movements
to be quantified (TruScan; Coulborn Instruments, Allentown, PA).
The next morning, dialysate samples were collected at 20-min inter-
vals. Samples were immediately assayed for DA and 5-HT by micro-
bore HPLC with electrochemical detection (ECD) as described pre-
viously (Baumann et al., 2000). Three baseline samples, which
differed by less than 10%, were collected, averaged, and all subse-
quent DA and 5-HT measures were expressed as a percentage of the
mean baseline. In vitro probe recoveries of DA and 5-HT ranged from
0.3 mg/kg/injection) or PAL-287 (0.001–0.3 mg/kg/injection) were
made available for self-administration. After a test session, monkeys
were returned to baseline conditions until cocaine- and saline-main-
tained responding were again stable. Doses were tested twice, once
the session after a cocaine baseline session and once the day after a
saline baseline session. In monkey AP01, results of the two test
sessions with dose of 0.03 mg/kg/injection PAL-287 varied widely.
Therefore, this dose was made available for nine consecutive sessions
until responding was stable.
Data Analysis. Raw data were analyzed as injections per session
for individual monkeys. Means of two sessions were calculated for
each dose of each drug, and the ranges of the two determinations
served as the measure of individual variability. When mean injec-
tions per session at a dose of a drug was above the mean for saline
sessions, and the ranges did not overlap, the dose of the drug was
considered to function as a positive reinforcer.
2
0 to 24%. Drug solutions were prepared immediately before use,
and doses are expressed as the salt. The motor activity of rats was
monitored during the dialysis sampling. Ambulation (i.e., forward
locomotion) and stereotypy (i.e., repetitive movements) were quan-
tified in 20-min bins and analyzed. The neurochemical and locomotor
data were evaluated statistically using one-factor (drug treatment)
repeated measures analysis of variance.
Neurotoxicity Studies
Rats received three sequential doses of PAL-287 (18 mg/kg i.p.),
(
ϩ)-methamphetamine (6.0 mg/kg i.p.), or (Ϯ)-MDMA (7.5 mg/kg
i.p.), one dose every 2 h. Vehicle-treated rats received saline injec-
tions (1 ml/kg i.p.) following the same schedule. Two weeks after the
injections, rats were sacrificed, brains were removed, and various
brain regions were dissected on ice. Cortical monoamine concentra-
tions were determined by HPLC with ECD as described previously
Procedures Carried Out at the Alcohol and Drug Abuse
Research Center, McLean Hospital
(
Baumann et al., 2001).
Subjects. Studies were conducted in a group of three male rhesus
monkeys implanted with chronic intravenous catheters under asep-
Assays of Human 5-HT Family Receptors
2
The activity of PAL-287 at cloned human 5-HT2A, 5-HT2B, and tic conditions. Monkeys weighing 8 to 12 kg and maintained on a diet
-HT2C receptors was conducted as described previously (Setola et of multiple vitamins, fresh fruit, and Lab Diet Jumbo Monkey bis-
5
al., 2003).
cuits (PMI Feeds, Inc., St. Louis, MO). In addition, monkeys could

1
364
Rothman et al.
receive 1 g of banana-flavored pellets (P. J. Noyes Co., Lancaster, dose-ranging studies in this procedure. A unit dose of 0.01 mg/kg/
NH) during daily sessions (see below). Water was continuously avail- injection cocaine was used because 1) it was the lowest dose to
able. A 12-h light/dark cycle was in effect (lights on from 7:00 AM to reliably maintain high rates of cocaine self-administration in all
7
:00 PM). The health of the monkeys was periodically monitored by monkeys, 2) we have shown previously that 0.01 mg/kg/injection
consulting veterinarians. Monkeys had visual, auditory, and olfac- cocaine and food maintain similar response rates in this procedure,
tory contact with other monkeys throughout the study. Operant and 3) previous studies have shown that behavior maintained by this
procedures and foraging toys provided opportunities for environmen- unit dose of cocaine is sensitive to the effects of pretreatment com-
tal manipulation and enrichment. Music or nature videotapes were pounds (Negus and Mello, 2002, 2003a). PAL-287 doses were tested
also played to provide additional environmental enrichment.
in an irregular order across monkeys. Treatment with saline and
Apparatus. Each monkey was housed individually in a well ven- each dose of PAL-287 was evaluated for 7 days. At the conclusion of
tilated stainless steel chamber (66 ϫ 76 ϫ 94 cm) equipped with a each 7-day treatment period, the maintenance dose of cocaine (0.032
custom-designed operant panel (28 ϫ 28 cm) mounted on the front mg/kg/injection) and saline control treatment was reinstated for at
wall. Three round translucent response keys (5.1 cm in diameter) least 4 days and until the number of reinforcers per day maintained
were arranged 2.54 cm apart in a horizontal row 3.2 cm from the top by cocaine and food returned to baseline levels. A suprathreshold
of the operant panel. Each key could be transilluminated by red or dose of 0.032 mg/kg/injection cocaine is used as a maintenance dose
green stimulus lights (Superbright LEDs). In addition, three circular during training and maintenance to ensure that a high-efficacy drug
translucent panels (1.9 cm in diameter) were located in a vertical reinforcer (and not merely a threshold reinforcer) is used during
column below the center response key and could be transilluminated
training and maintenance phases of the experiment. This interval
by red or green stimulus lights. Each housing chamber was also between successive treatments was designed to reduce the possibil-
equipped with a pellet dispenser (model G5210; Gerbrands, Arling- ity of carryover effects from one treatment condition to the next.
ton, MA) and two syringe pumps (model B5P-lE; Braintree Scientific,
Data Analysis. The primary dependent variables were the num-
Braintree, MA; or model 980210, Harvard Apparatus Inc., South ber of cocaine injections (maximum 80 per day) and food pellets
Natick, MA), one for each lumen of the double lumen catheter. One (maximum 100 per day) delivered per day. Data from the last 3 days
syringe pump (the self-administration pump) was used to deliver of each 7-day treatment were used for data analysis. Effects of
cocaine injections. The second syringe pump (the treatment pump) PAL-287 on cocaine- and food-maintained responding were ex-
was used to deliver saline or test drug as described below for each
pressed as the percentage of control levels of cocaine- and food-
procedure. The treatment pump delivered injections every 20 min maintained responding observed during saline treatment. Data were
from 10:30 AM each day until 9:30 AM the next morning for a total
of 3 injections/h and 69 injections/day. No treatment injections were
delivered between 9:30 AM and 10:30 AM; and during this period,
analyzed by two-way ANOVA, with type of reinforcer (cocaine or
food) and dose of PAL-287 (0.1–1.0 mg/kg/h) as the two factors. A
significant ANOVA was followed by the Newman-Keuls post hoc test.
monkeys received their morning ration of food, and their health ED50 values for PAL-287 were also determined for each monkey,
status was evaluated by the technical staff. Operation of the operant with the ED50 defined as the dose of PAL-287 that reduced cocaine-
panels and data collection were accomplished with microprocessors or food-maintained responding to 50% of control. A mean Ϯ S.E.M.
and software purchased from MED Associates (Georgia, VT).
ED50 value was then determined, and ED50 values for PAL-287 to
Second-Order Schedule of Cocaine and Food Availability. reduce cocaine- and food-maintained responding were compared by t
The effects of chronic PAL-287 administration on cocaine- and food- test. For all statistical tests, the criterion for significance was set a
maintained responding under a second-order schedule were exam- priori at P Ͻ 0.05.
ined using procedures identical to those used in our previous studies
of the effects of d-amphetamine treatment (Negus and Mello, 2003).
Alternating daily sessions of food and cocaine availability were as-
sociated with different colored stimulus lights projected on the center
response key of the operant response panel. Specifically, red stimu-
lus lights signaled food availability and green stimulus lights sig-
naled the availability of cocaine injections (delivered in a volume of
Results
More than 350 compounds were screened for activity as
substrate-type releasing agents at DA transporters (DATs),
-HT transporters (SERTs), and norepinephrine transport-
5
0
.1 ml in 1 s). Under the terminal schedule, the completion of a ers (NETs). These efforts identified PAL-287 as a compound
variable ratio of 16 responses on the center response key resulted in different from amphetamine that has potent actions in vitro
the illumination for 1 s of an appropriately colored stimulus light as a substrate for DATs, SERTs, and NETs (Fig. 1). The EC50
(
red for food, green for drug) underneath the center key (VR16:S). In
values of PAL-287 at DAT (12.6 Ϯ 0.4 nM) and NET (11.1 Ϯ
.9 nM) are similar to that reported for (ϩ)-methamphet-
addition, completion of this VR response requirement a fixed ratio of
two times (FR2) resulted in delivery of the available reinforcer and
the initiation of a 10-s time-out period, during which the stimulus
light illuminating the center response key was turned off and re-
sponding had no scheduled consequences. This terminal second-
order schedule is designated as FR2(VR16:S). The two side keys were
not transilluminated during sessions of food and cocaine availability,
and responding on these keys had no scheduled consequences. Four
0
amine, 24.5 Ϯ 2.1 and 12.3 nM, respectively. However, the
EC50 value of PAL-287 at SERT (3.4 Ϯ 0.2 nM) is much lower
than that of (ϩ)-methamphetamine (736 Ϯ 45 nM) and am-
phetamine (1765 Ϯ 94 nM) and is about 10-fold lower than
reported for 5-HT (44.4 Ϯ 5.3 nM) (Rothman et al., 2001).
As depicted in Fig. 2, in vivo microdialysis experiments
food sessions and four drug sessions were conducted during each demonstrated that i.v. administration of PAL-287 increased
experimental day. Food sessions began at 6:00 AM, 11:00 AM, 3:00 extracellular DA and 5-HT in rat prefrontal cortex in a dose-
PM, and 7:00 PM, and drug sessions began at 7:00 AM, 12:00 PM,
dependent manner. PAL-287 elevated 5-HT to a greater ex-
4
:00 PM, and 8:00 PM. At all other times, responding had no sched-
tent than DA at both doses tested. Figure 3 shows that i.v.
ϩ)-amphetamine increased extracellular DA with minimal
effects on 5-HT. It is noteworthy that 3 mg/kg PAL-287 and
mg/kg (ϩ)-amphetamine increased extracellular DA to sim-
ilar extent, about 8-fold above baseline. Despite the large
elevations in extracellular DA produced by PAL-287, the
uled consequences. Each food and drug session lasted 1 h or until 25
food pellets or 20 injections had been delivered, whichever occurred
first. Thus, monkeys could earn a maximum of 100 food pellets per
day and 80 injections/day. Studies were conducted 7 days/week.
Testing Procedures. The effects of treatment with chronic saline
and PAL-287 (0.1–1.0 mg/kg/h) on food- and cocaine-maintained
(
1
responding were compared during availability of 0.01 mg/kg/injec- 3-mg/kg dose of the drug increased ambulation to a much
tion cocaine. These doses of PAL-287 were based on preliminary lower level compared with 1 mg/kg (ϩ)-amphetamine (com-

Releasing Agents Suppress Cocaine Self-Administration
1365
Fig. 3. Dose-response effects of (ϩ)-amphetamine on extracellular DA
and 5-HT in rat prefrontal cortex as determined by in vivo microdialysis.
Rats received i.v. injection of 0.3 mg/kg (ϩ)-amphetamine at time 0,
followed by 1 mg/kg 60 min later. Data are mean Ϯ S.E.M. for seven rats
per group, expressed as percentage of baseline. Baseline levels of DA and
Fig. 1. Dose-response curves showing PAL-287-induced release of
3
[
H]neurotransmitter via the DAT, NET, and SERT. The curves shown
5
-HT were 0.38 Ϯ 0.07 and 0.24 Ϯ 0.06 pg/5 ␮l. ءء, P Ͻ 0.05 compared
are one representative experiment. The data of three experiments were
pooled and fit to the two-parameter logistic equation for the best fit
estimate of the EC50 (nanomolar Ϯ S.D.), reported in the text. The
structure of PAL-287 is shown in the inset.
with preinjection control, Duncan’s post hoc test.
Fig. 2. Dose-response effects of PAL-287 on extracellular DA and 5-HT in
rat prefrontal cortex as determined by in vivo microdialysis. Rats re-
ceived i.v. injection of 1 mg/kg PAL-287 at time 0, followed by 3 mg/kg 60
min later. Data are mean Ϯ S.E.M. for seven rats per group, expressed as
percentage of baseline. Baseline levels of DA and 5-HT were 0.43 Ϯ 0.07
and 0.27 Ϯ 0.06 pg/5 ␮l. ءء, P Ͻ 0.05 compared with preinjection control,
Duncan’s post hoc test.
Fig. 4. Dose-response effects of PAL-287 on ambulation and stereotypy in
rats undergoing microdialysis sampling. Rats received i.v. injections of 1
mg/kg PAL-287 at time 0, followed by 3 mg/kg 60 min later. Data are
mean Ϯ S.E.M. for seven rats per group, expressed as distance traveled
in centimeters (ambulation) and number of repetitive movements (ste-
reotypy). ءء, P Ͻ 0.05 compared with preinjection control, Duncan’s post
hoc test.
pare Figs. 4 and 5). Thus, PAL-287 displayed very weak
locomotor stimulant activity.
The ability of PAL-287 to induce long-term depletion of
brain 5-HT (i.e., neurotoxicity) was determined by adminis-
tration of three sequential doses (18 mg/kg i.p.) every 2 h. As
positive controls, other rats received (ϩ)-methamphetamine
monkeys self-administered PAL-287 when it was substituted
for cocaine, whereas the other three did not.
Rhesus monkeys were trained to respond for food and
cocaine injections as detailed previously (Negus and Mello,
2003b). Figure 8 shows the effects of chronic, 7-day treatment
with PAL-287 on cocaine- and food-maintained responding in
three rhesus monkeys. During saline treatment, the mean
number Ϯ S.E.M. of cocaine injections per day was 79.4 Ϯ 0.6
(
6.0 mg/kg i.p.) or (Ϯ)-MDMA (7.5 mg/kg i.p.) administered
according to the same schedule. Rats were sacrificed 2 weeks
later and cortical 5-HT levels determined as described under
Materials and Methods. The results (Fig. 6) indicated that
whereas both (ϩ)-methamphetamine and MDMA decreased
cortical 5-HT, PAL-287 did not. None of the treatments de-
pleted cortical DA (data not shown).
(
maximum of 80), and the mean number of food pellets per
day was 96.1 Ϯ 3.9 (maximum of 100). PAL-287 produced a
dose-dependent decrease in both cocaine- and food-main-
tained responding, and statistical results are reported in the
figure legend. A dose of 1.0 mg/kg/h PAL-287 significantly
Using published methods (Wee et al., 2004), four rhesus
monkeys were trained to self-administer cocaine. As reported reduced both cocaine- and food-maintained responding; how-
in Fig. 7, the cocaine dose-response curves in the monkeys ever, the suppression of cocaine self-administration was
followed a typical “inverted U” shape. PAL-287 (0.001–0.3 greater than the reduction in food-maintained responding.
mg/kg/injection) displayed little or no reinforcing effect, in- The ED50 values for PAL-287 to reduce cocaine- and food-
dicative of a compound with low abuse potential. One of four maintained responding were 0.49 Ϯ 0.04 and 0.85 Ϯ 0.17

1
366
Rothman et al.
Fig. 7. Self-administration of cocaine and PAL-287 by rhesus monkeys.
Drugs were available under a FR 25 schedule of reinforcement for 2
h/day. Each point is the mean of two sessions of access to each dose of the
drugs. Data are mean Ϯ S.E.M. for n ϭ 4 monkeys. Symbols without bars
have variability smaller than the points. ء, P Ͻ 0.05 compared with
saline-injected control, Duncan’s post hoc test.
Fig. 5. Dose-response effects of (ϩ)-amphetamine on ambulation and
stereotypy in rats undergoing microdialysis sampling. Rats received i.v.
injections of 0.3 mg/kg (ϩ)-amphetamine at time 0, followed by 1 mg/kg
60 min later. Data are mean Ϯ S.E.M. for seven rats per group, expressed
as distance traveled in centimeters (ambulation) and number of repeti-
tive movements (stereotypy). ء, P Ͻ 0.05 compared with preinjection
control, Duncan’s post hoc test.
Fig. 8. Effects of chronic, 7-day treatment with PAL-287 on cocaine- and
food-maintained responding. Abscissa, dose PAL-287 in milligrams per
kilogram per hour (log scale). Ordinate, percentage of control levels of
cocaine- and food-maintained responding. Control values were defined as
levels of cocaine- or food-maintained responding observed during 7 days
of saline treatment (79.4 Ϯ 0.6 cocaine injections per day of a maximum
of 80; 96.3 Ϯ 3.9 food pellets per day of a maximum of 100). Each point
shows mean data Ϯ S.E.M. for three monkeys collected during the last 3
days of each 7-day treatment. Two-way ANOVA indicated a significant
effect of PAL-287 dose [F(2,4) ϭ 167; P ϭ 0.0001) but not a significant
effect of reinforcer type [F(2,2) ϭ 7.26; P ϭ 0.114) or a significant inter-
action [F(2,4) ϭ 3.86; P ϭ 0.116). Post hoc analysis was conducted with
Fig. 6. Effects of high-dose administration of PAL-287 on long-term 5-HT
depletion in rat brain. Rats received three sequential i.p. injections of
PAL-287 (18 mg/kg), (ϩ)-methamphetamine (METH; 6.0 mg/kg), MDMA
(
7.5 mg/kg), or saline, one injection every 2 h. Rats were sacrificed 2
weeks after injections, and brain tissue levels of 5-HT and 5-HIAA were
determined by HPLC with ECD. Data are mean Ϯ S.E.M. for n ϭ 6 rat
per group, expressed as percentage of baseline. ء, P Ͻ 0.05 compared with
saline-injected control, Duncan’s post hoc test.
mg/kg/h, respectively, and these values were not significantly the Newman-Keuls test. ء, significant effect of PAL-287 dose in compar-
different (P ϭ 0.19).
We also evaluated the ability of PAL-287 to activate cloned
human 5-HT2A, 5-HT2B, and 5-HT2C receptors (for details,
see Setola et al., 2003). The results (Table 1) indicated that
PAL-287 is a full agonist at the 5-HT2B receptor (EC50 ϭ 40
nM) and the 5-HT2A receptor (EC50 ϭ 466 nM). It is a potent
ison with control for a given reinforcer, P Ͻ 0.05. [yen], food-maintained
responding was significantly greater than cocaine-maintained respond-
ing at that dose of PAL-287, P Ͻ 0.05.
Discussion
The main purpose of the present study was to identify a
partial 5-HT2C agonist (EC50 ϭ 2.3 nM, Emax ϭ 20%). The novel monoamine releaser that could be developed as an
partial agonist action of PAL-287 at the 5-HT2C receptor agonist replacement medication for treating stimulant de-
predicts possible anorectic actions of PAL-287 (Vickers et al., pendence. We chose to focus our attention on a nonamphet-
1
999) and may also reduce the rewarding effects produced by amine molecule with dual DA/5-HT releasing properties for
the DA releasing actions of PAL-287 (for review, see Czoty, et several reasons. First, a wealth of information supports the
al., 2002). The relatively weak potency at 5-HT2A and 5-HT2B existence of a dual deficit in synaptic DA and 5-HT during
receptors, compared with its activity at SERT, suggest that withdrawal from chronic stimulant abuse. A medicine that
PAL-287 will have minimal functional actions at 5-HT2A and releases DA and 5-HT might alleviate stimulant withdrawal
5
-HT2B receptors in vivo.
symptoms by restoring neurochemical deficits in abstinent

Releasing Agents Suppress Cocaine Self-Administration
1367
TABLE 1
Summary of studies conducted with cloned human serotonin receptors
h5-HT2A
h5-HT2B
h5-HT2Ca
h5-HT2C (VGI)b
5
-HT
EC50 (nM) (pEC50 Ϯ S.D.)
Emax (%)
13 (1.13 Ϯ 0.08)
2.5 (0.39 Ϯ 0.19)
1.2 (0.07 Ϯ 0.18)
2.0 (0.29 Ϯ 0.1)
100
100
100
100
PAL-287
EC50 (nM) (pEC50 Ϯ S.D.)
Emax (%)
466 (2.7 Ϯ 0.08)
40 (1.6 Ϯ 0.2)
2.3 (0.36 Ϯ 0.44)
102 (2.01 Ϯ 0.20)
74
91
20
35
a
b
Studies measured drug effects on phosphoinositide hydrolysis.
Refers to mRNA editing program. Each value is the mean Ϯ S.D. (n ϭ 3).
addicts. Second, DA releasers (e.g., phentermine) and 5-HT this effect was not selective. The significant decreases in
releasers (e.g., fenfluramine) are both known to suppress food-maintained responding suggest that decreases in co-
ongoing stimulant self-administration in animal models. By caine self-administration may have resulted, at least in part,
designing a single molecular entity with mixed DA/5-HT from nonselective effects of PAL-287 that limited the ability
releasing activity, two separate mechanisms could be re- of the monkeys to emit operant responses. It should be noted,
cruited to suppress illicit stimulant use. Finally, we predicted however, that cocaine self-administration was virtually elim-
that a dual DA/5-HT releaser would have reduced abuse inated by 1.0 mg/kg/h PAL-287, whereas food-maintained
liability compared with a selective DA releaser, since 5-HT was decreased by 65%, and cocaine self-administration was
release can dampen the stimulant effects mediated by DA. decreased more than food-maintained responding in all three
Our results with PAL-287 confirm the hypothesis that a monkeys. These results demonstrate that chronic, 7-day
nonamphetamine substrate at DATs and SERTs will release treatment with PAL-287 produced a dose-dependent and
DA and 5-HT from neurons in vivo and thereby suppress moderately selective decrease in cocaine self-administration
ongoing cocaine self-administration. PAL-287 displays a in rhesus monkeys responding under a second-order sched-
number of desirable qualities for a candidate medication, ule. Future studies should examine the effect of PAL-287 on
including minimal locomotor activation, lack of long-term other unit doses of cocaine.
neurotoxicity, and low abuse potential.
Our findings with PAL-287 in monkeys are reminiscent of
Preclinical and human data suggest that withdrawal from the suppression of cocaine self-administration produced by
chronic cocaine is associated with impairments in 5-HT neu- (ϩ)-amphetamine reported previously, although amphet-
ronal function, in addition to the well accepted deficits in DA amine displays greater selectivity than PAL-287 in reducing
function (Dackis and Gold, 1985). Perhaps the most compel- cocaine self-administration as opposed to food-maintained
ling evidence for 5-HT deficits in cocaine addiction is the responding (Negus and Mello, 2003b). Grabowski et al. (2001,
occurrence of a psychiatric syndrome resembling major de- 2004) showed that a slow-release formulation of (ϩ)-amphet-
pression after abstinence from binge cocaine use (Dackis and amine is effective in maintaining cocaine addicts in treat-
Gold, 1985; Gawin and Kleber, 1986), coupled with the in- ment and reducing illicit cocaine use. We predict that agents
creased prevalence of suicidal ideation and suicide attempts like PAL-287, which have mixed DA/5-HT releasing activity,
among cocaine addicts (Garlow et al., 2003). The well estab- will possess the therapeutic effects of amphetamine-type
lished role of 5-HT dysfunction in mediating depression and monoamine releasers, while minimizing the adverse effects
suicide (for review, see Mann, 2003) suggests that decreased associated the phenethylamine structure. The role of NE in
synaptic 5-HT might play a role in cocaine withdrawal states. the actions of PAL-287 is an important issue awaiting addi-
In support of this notion, rats withdrawn from chronic co- tional study (Rothman et al., 2001).
caine treatment display abnormalities in 5-HT-mediated
It is interesting to note that PAL-287 and PAL-313 (para-
neuroendocrine secretion that are similar to those observed methylamphetamine), the reinforcing effects of which were
in humans diagnosed with major depression (Baumann and reported in a recent article (Wee et al., 2005), are both equi-
Rothman, 1998).
potent as releasers across the monoamines. Yet PAL-287 was
We have previously proposed a dual deficit model of stim- not a reinforcer under a simple FR10 schedule, whereas
ulant withdrawal where drug-induced decreases in synaptic PAL-313 was a reinforcer under a progressive ratio schedule
DA and 5-HT contribute to withdrawal dysphoria, drug crav- where the response requirement was increased to at least
ing, and relapse (for review, see Baumann et al., 2000). 200 lever presses per injection. Additional research will be
Initial observations in human patients receiving the DA re- required to determine why these two agents differ in their
leaser phentermine and the 5-HT releaser fenfluramine as reinforcing efficacy.
medications, showed that concurrent DA and 5-HT release
One limitation of using amphetamine-type drugs as med-
can ameliorate symptoms of cocaine withdrawal and reduce ications is their high abuse potential, which is related to
illicit cocaine use (Rothman et al., 1994; Kampman et al., stimulation of DA release in mesolimbic reward pathways.
2
000). These preliminary clinical findings strengthened the Several lines of evidence support the hypothesis that eleva-
rationale for development of DAT and SERT substrates as tions in synaptic 5-HT can counteract the stimulant and
potential treatments for stimulant addiction. reinforcing effects mediated by elevations in synaptic DA
The present data with PAL-287 support the use of mono- (Daw et al., 2002; Burmeister et al., 2004). For example,
amine releasers as agonist-substitution medications for the 5-HT precursor loading with dietary tryptophan attenuates
treatment of stimulant addictions. A dose of 1.0 mg/kg/h amphetamine self-administration in rats (Smith et al., 1986).
PAL-287 virtually eliminated cocaine self-administration in Other studies show that pretreatment with 5-HT reuptake
rhesus monkeys by the end of the 7-day treatment, although inhibitors reduces intravenous cocaine self-administration in

1
368
Rothman et al.
rats (Carroll et al., 1990) and squirrel monkeys (Howell and expression level) that are not characteristic of native tissue
Byrd, 1995). Consistent with these data, cocaine analogs that (Bockaert et al., 1997). Although our data suggest that PAL-
have enhanced 5-HT transporter affinity (Roberts et al., 287 will not significantly activate 5-HT2B and 5-HT2A recep-
1
999) support less self-administration behavior than analogs tors in vivo, further studies will be needed to address this
with lower 5-HT transporter affinity. The comparative mi- question more directly.
crodialysis results reported here for PAL-287 and (ϩ)-am-
The use of stimulant-like medications to treat stimulant
phetamine provide direct support for the hypothesis that addictions is an approach described as “agonist substitution”
5
-HT release dampens stimulant effects mediated by DA therapy. This strategy involves administering medications
release. PAL-287 (3 mg/kg i.v.) and (ϩ)-amphetamine (1 that are less potent and less addictive than cocaine or meth-
mg/kg i.v.) increased extracellular DA to a similar extent, but amphetamine but that nevertheless decrease stimulant
the locomotor stimulant effects of PAL-287 were much less abuse because of shared neurochemical properties with the
than those of (ϩ)-amphetamine. We believe that reduced illicit drugs (Gorelick, 1998). Viewed from this perspective,
locomotor activation afforded by PAL-287 is related to the agonist substitution therapy can be described as “neuro-
marked increase in extracellular 5-HT produced by this drug, chemical normalization” therapy—the treatment medication
an effect which is not shared by the selective DA releaser “normalizes” dysregulated neurochemistry by substituting
(
ϩ)-amphetamine. for the abused drug. This approach has been explored for
A number of additional side effects could limit the use of the treatment of cocaine dependence (Alim et al., 1995;
dual DA/5-HT releasing agents. For example, some 5-HT Grabowski et al., 1997, 2001; Kampman et al., 2000; Walsh et
releasing agents cause adverse effects that include neurotox- al., 2000). Our results with PAL-287 extend the concept of
icity, cardiac valvulopathy, and primary pulmonary hyper- agonist substitution by using a dual DA/5-HT releaser that
tension (PPH) (for review, see Rothman and Baumann, exhibits the desired therapeutic effects of a replacement
2
002). Our recent investigations support the possibility of medication without the adverse side effects associated with
developing dual DA/5-HT releasers that lack adverse effects. prototypical psychomotor stimulants.
In particular, it was suggested that a lead drug molecule
In summary, the findings reported here demonstrate that
should be chemically distinct from the phenethylamine struc- a nonamphetamine monoamine releasing agent with low
ture shared by amphetamine-like agents because substituted abuse potential and minimal neurotoxicity can suppress co-
phenethylamines are associated with neurotoxicity in ani- caine self-administration in subhuman primates. As such,
mals. Furthermore, the candidate medication should lack PAL-287 represents the prototype for a new generation of
significant interaction at the 5-HT2B receptor, a site that is drugs that enhance biogenic amine release by acting as sub-
implicated in pathogenesis of fenfluramine-associated car- strates at multiple biogenic amine transporters (DATs,
diac valvulopathy (Fitzgerald et al., 2000; Rothman et al., SERTs, and NETs). It seems possible that drugs with a
2
000; Setola et al., 2003).
similar mode of action will provide neurochemical normaliza-
The term “5-HT neurotoxicity”, when used in the present tion therapy for the treatment of cocaine addiction and also
context, refers to high-dose administration of selective 5-HT be potentially useful for treating depression, obsessive com-
releasers (e.g., fenfluramine) often causing persistent deple- pulsive disorder, attention deficit disorder, and obesity.
tion of brain tissue 5-HT and 5-HT transporters. A key ob-
servation is that not all SERT substrates deplete 5-HT, since
repeated administration of the SERT substrate m-chlorophe-
nylpiperazine (mCPP) fails to deplete brain 5-HT, despite
producing elevations of extracellular 5-HT comparable with
Acknowledgments
We acknowledge the expert technical assistance of John Partilla
and Robert Clark.
fenfluramine (Baumann et al., 2001). Like mCPP, PAL-287
produces elevations in extracellular 5-HT but does not pro-
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Address correspondence to: Dr. Richard B. Rothman, Clinical Psychophar-
macology Section, Intramural Research Program, National Institute on Drug
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Baltimore, MD 21224. E-mail: rrothman@intra.nida.nih.gov
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