meso-Transdiene analogs inhibit vesicular monoamine transporter-2 function and methamphetamine-evoked dopamine release.
ABSTRACT Lobeline, a nicotinic receptor antagonist and neurotransmitter transporter inhibitor, is a candidate pharmacotherapy for methamphetamine abuse. meso-Transdiene (MTD), a lobeline analog, lacks nicotinic receptor affinity, retains affinity for vesicular monoamine transporter 2 (VMAT2), and, surprisingly, has enhanced affinity for dopamine (DA) and serotonin transporters [DA transporter (DAT) and serotonin transporter (SERT), respectively]. In the current study, MTD was evaluated for its ability to decrease methamphetamine self-administration in rats relative to food-maintained responding. MTD specifically decreased methamphetamine self-administration, extending our previous work. Classical structure-activity relationships revealed that more conformationally restricted MTD analogs enhanced VMAT2 selectivity and drug likeness, whereas affinity at the dihydrotetrabenazine binding and DA uptake sites on VMAT2 was not altered. Generally, MTD analogs exhibited 50- to 1000-fold lower affinity for DAT and were equipotent or had 10-fold higher affinity for SERT, compared with MTD. Representative analogs from the series potently and competitively inhibited [(3)H]DA uptake at VMAT2. (3Z,5Z)-3,5-bis(2,4-dichlorobenzylidene)-1-methylpiperidine (UKMH-106), the 3Z,5Z-2,4-dichlorophenyl MTD analog, had improved selectivity for VMAT2 over DAT and importantly inhibited methamphetamine-evoked DA release from striatal slices. In contrast, (3Z,5E)-3,5-bis(2,4-dichlorobenzylidene)-1-methylpiperidine (UKMH-105), the 3Z,5E-geometrical isomer, inhibited DA uptake at VMAT2, but did not inhibit methamphetamine-evoked DA release. Taken together, these results suggest that these geometrical isomers interact at alternate sites on VMAT2, which are associated with distinct pharmacophores. Thus, structural modification of the MTD molecule resulted in analogs exhibiting improved drug likeness and improved selectivity for VMAT2, as well as the ability to decrease methamphetamine-evoked DA release, supporting the further evaluation of these analogs as treatments for methamphetamine abuse.
- SourceAvailable from: Veronica M Chiu[Show abstract] [Hide abstract]
ABSTRACT: Addiction to methamphetamine (METH) is thought to be mediated by dopaminergic effects in the reward pathway in the brain via the A10 dopaminergic pathway. Herein we describe an overview of the results of the basic preclinical science undertaken to provide mechanistic insights into the action of amphetamines in general and METH in particular. A brief history of amphetamine and METH use and abuse is given, and an overview of the relevant chemical aspects of amphetamine as they relate to neurotransmitters in general is made. A review of the methods used to study the biochemical effects of METH is outlined. Finally, a focused analysis of the kinetic mechanisms of action of the amphetamines in general, and METH in particular, at the transmembrane transporters and at the intracellular vesicular storage sites is made. A description of how catecholaminergic and serotonergic nerve signaling may be altered by METH is proposed. Overall, the emphasis here is on differences in effects observed between the striatal (the A9 substantia nigral dopamine pathway) and nucleus accumbens (the A10, ventral tegmental pathway) areas of the brain following acute as well as repeated dosing and withdrawal.Current Drug Abuse Reviews 09/2012; 5:227-242.
- [Show abstract] [Hide abstract]
ABSTRACT: Previous research suggests that the vesicular monoamine transporter-2 (VMAT2) is a novel target for the treatment of methamphetamine (METH) abuse. The effects GZ-793A, a novel, selective, and potent lobelane analog, on the rewarding effects of METH, cocaine, and palatable food in rats were determined. GZ-793A (3-30 mg/kg, s.c.) was administered 20 min prior to each session in which the groups of rats pressed a lever for infusions of METH (0.03 mg/kg/infusion), cocaine (0.3 mg/kg/infusion), or food pellets. Tolerance to repeated GZ-793A (15 mg/kg, s.c. for 7 days) on METH self-administration and food-maintained responding was determined. The ability of increasing doses of METH (0.001-0.56 mg/kg, i.v.) to surmount inhibition produced by GZ-793A (15 mg/kg, s.c.) was determined. Self-administration of GZ-793A (0.01-0.3 mg/kg/infusion, i.v.) was tested as a substitute for METH infusion. GZ-793A (15 mg/kg, s.c.) was administered 20 min prior to METH or saline conditioning in a place preference test. GZ-793A specifically decreased METH self-administration, without the development of tolerance. Increasing the unit dose of METH did not surmount the inhibition produced by GZ-793A on METH self-administration. GZ-793A did not serve as a substitute for self-administered METH. GZ-793A blocked METH-induced conditioned place preference (CPP) and did not induce CPP alone. These results indicate that VMAT2 is a viable target for pharmacological inhibition of METH reward and that GZ-793A represents a new lead in the discovery of a treatment for METH abuse.Psychopharmacology 09/2011; 220(2):395-403. · 4.06 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Glutamate contributes to the reinforcing and stimulant effects of methamphetamine, yet its potential role in the interoceptive stimulus properties of methamphetamine is unknown. In this study, adult male Sprague-Dawley rats were trained to discriminate methamphetamine [1.0 mg/kg, intraperitoneally] from saline in a standard operant discrimination task. The effects of methamphetamine (0.1-1.0 mg/kg, intraperitoneally); N-methyl-D-aspartate (NMDA) receptor channel blockers, MK-801 (0.03-0.3 mg/kg, intraperitoneally) and ketamine (1.0-10.0 mg/kg, intraperitoneally); polyamine site NMDA receptor antagonist, ifenprodil (1-10 mg/kg); α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (1-10 mg/kg, intraperitoneally); and metabotropic 5 glutamate receptor antagonist, 6-methyl-2-(phenylethynyl)pyridine (1-10 mg/kg), given alone were determined in substitution tests. The effects of MK-801 (0.03 and 0.1 mg/kg), ketamine (1.0 and 3.0 mg/kg), ifenprodil (5.6 mg/kg), 6-cyano-7-nitroquinoxaline-2,3-dione (5.6 mg/kg), and 6-methyl-2-(phenylethynyl)pyridine (5.6 mg/kg) were also tested in combination with methamphetamine to assess for alterations in the methamphetamine cue. In substitution tests, none of the test drugs generalized to the methamphetamine cue. However, ketamine and ifenprodil produced significant leftward shifts in the methamphetamine dose-response curve. In addition, the potention by MK-801 nearly attained significance. These results suggest that blockade of the NMDA receptor augments the interoceptive stimulus properties of methamphetamine.Behavioural pharmacology 09/2011; 22(5-6):516-24. · 2.85 Impact Factor
meso-Transdiene Analogs Inhibit Vesicular Monoamine
Transporter-2 Function and Methamphetamine-Evoked
David B. Horton, Kiran B. Siripurapu, Seth D. Norrholm, John P. Culver,
Marhaba Hojahmat, Joshua S. Beckmann, Steven B. Harrod,1Agripina G. Deaciuc,
Michael T. Bardo, Peter A. Crooks, and Linda P. Dwoskin
Department of Pharmaceutical Sciences, College of Pharmacy (D.B.H., K.B.S., S.D.N., J.P.C., M.H., A.G.D., P.A.C., L.P.D.),
and Department of Psychology, College of Arts and Sciences (J.S.B., S.B.H., M.T.B.), University of Kentucky,
Received September 13, 2010; accepted December 16, 2010
Lobeline, a nicotinic receptor antagonist and neurotransmitter
transporter inhibitor, is a candidate pharmacotherapy for meth-
amphetamine abuse. meso-Transdiene (MTD), a lobeline ana-
log, lacks nicotinic receptor affinity, retains affinity for vesicular
monoamine transporter 2 (VMAT2), and, surprisingly, has en-
hanced affinity for dopamine (DA) and serotonin transporters
[DA transporter (DAT) and serotonin transporter (SERT), re-
spectively]. In the current study, MTD was evaluated for its
ability to decrease methamphetamine self-administration in
rats relative to food-maintained responding. MTD specifically
decreased methamphetamine self-administration, extending
our previous work. Classical structure-activity relationships re-
vealed that more conformationally restricted MTD analogs en-
hanced VMAT2 selectivity and drug likeness, whereas affinity at
the dihydrotetrabenazine binding and DA uptake sites on
VMAT2 was not altered. Generally, MTD analogs exhibited 50-
to 1000-fold lower affinity for DAT and were equipotent or had
10-fold higher affinity for SERT, compared with MTD. Repre-
sentative analogs from the series potently and competitively
inhibited [3H]DA uptake at VMAT2. (3Z,5Z)-3,5-bis(2,4-dichlo-
robenzylidene)-1-methylpiperidine (UKMH-106), the 3Z,5Z-2,4-
dichlorophenyl MTD analog, had improved selectivity for
VMAT2 over DAT and importantly inhibited methamphetamine-
evoked DA release from striatal slices. In contrast, (3Z,5E)-3,5-
the 3Z,5E-geometrical isomer, inhibited DA uptake at VMAT2,
but did not inhibit methamphetamine-evoked DA release.
Taken together, these results suggest that these geometrical
isomers interact at alternate sites on VMAT2, which are asso-
ciated with distinct pharmacophores. Thus, structural modifi-
cation of the MTD molecule resulted in analogs exhibiting im-
proved drug likeness and improved selectivity for VMAT2, as
well as the ability to decrease methamphetamine-evoked DA
release, supporting the further evaluation of these analogs as
treatments for methamphetamine abuse.
Methamphetamine abuse is a serious public health con-
cern (Substance Abuse and Mental Health Services Admin-
istration, Office of Applied Studies, 2008). Pharmacothera-
pies are not available to treat methamphetamine abuse.
Efforts have focused on the dopamine (DA) transporter
(DAT) as a therapeutic target (Dar et al., 2005; Howell et al.,
2007; Tanda et al., 2009), because methamphetamine inter-
acts with DAT to increase extracellular DA concentrations,
leading to its reinforcing properties (Wise and Bozarth, 1987;
Di Chiara and Imperato, 1988; Di Chiara et al., 2004). DAT
translocates DA from the extracellular space into presynap-
tic terminals, whereas methamphetamine reverses DAT
translocation to increase DA extracellularly (Fischer and
Cho, 1979; Liang and Rutledge, 1982; Sulzer et al., 1995).
The above approach has not led to therapeutic agents for
methamphetamine abuse, although several DAT inhibitors
currently are undergoing clinical trials.
A largely unexplored target of methamphetamine action is
vesicular monoamine transporter 2 (VMAT2). By interacting
with VMAT2, methamphetamine increases cytosolic DA con-
centrations available for translocation by DAT to the extra-
This work was supported by the National Institutes of Health National
Institute on Drug Abuse [Grants DA 13519, DA 016176, DA 021287].
The University of Kentucky holds patents on the analogs described, and
some of the patents have been licensed by Yaupon Therapeutics, Inc. A poten-
tial royalty stream to L.P.D. and P.A.C. may occur consistent with University
of Kentucky policy. Both L.P.D. and P.A.C. are founders of, and have financial
interest in, Yaupon Therapeutics, Inc.
1Current affiliation: Department of Psychology, Behavioral Neuroscience
Program, University of South Carolina, Columbia, South Carolina.
Article, publication date, and citation information can be found at
S The online version of this article (available at http://jpet.aspetjournals.org)
contains supplemental material.
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
JPET 336:940–951, 2011
Vol. 336, No. 3
Printed in U.S.A.
cellular compartment (Sulzer and Rayport, 1990; Pifl et al.,
1995; Sulzer et al., 1995). Current research focuses on the
discovery of novel compounds that interact with VMAT2 and
inhibit the pharmacological effects of methamphetamine. Lo-
beline, the major alkaloid of Lobelia inflata, inhibits VMAT2
function (Teng et al., 1997, 1998), has high affinity for [3H]di-
hydrotetrabenazine (DTBZ) binding sites on VMAT2 (Kil-
bourn et al., 1995; Miller et al., 2004), and decreases amphet-
amine-evoked DA release from rat striatal slices (Miller et
al., 2001). However, lobeline is not selective for VMAT2,
acting as a nicotinic acetylcholine receptor (nAChR) antago-
nist with low affinity for DAT and the serotonin transporter
(SERT) (Damaj et al., 1997; Flammia et al., 1999; Miller et
al., 2000, 2004). Lobeline also decreases methamphetamine-
induced hyperactivity, behavioral sensitization, and self-ad-
ministration in rats (Harrod et al., 2001; Miller et al., 2001).
It is noteworthy that lobeline is not self-administered, indi-
cating lack of abuse liability (Harrod et al., 2003). Based on
these preclinical findings, lobeline is being evaluated as a
treatment for methamphetamine abuse. Initial-phase clini-
cal trials report that lobeline is safe in methamphetamine
Lobeline has a central piperidine ring with phenyl rings
attached at C-2 and C-6 of the piperidine ring by ethylene
linkers containing hydroxyl and keto functionalities at the
C8 and C10 positions on the linkers, respectively (Fig. 1).
Potency and selectivity for VMAT2 were improved based on
structure-activity relationships (SARs), with the emergence
of two new lead compounds, i.e., lobelane and meso-trans-
diene (MTD) (Zheng et al., 2005; Nickell et al., 2010a). Lo-
belane is a lobeline analog with defunctionalized (hydroxyl
and keto groups of lobeline eliminated from the linkers) and
saturated linkers. Subsequent reports described the preclin-
ical evaluation of lobelane as well as analogs based on the
lobelane structural scaffold (Beckmann et al., 2010; Nickell
et al., 2010b). MTD is a lobeline analog with defunctionalized
and unsaturated (double bonds) linkers (Fig. 1). Compared
with lobeline, MTD was found to exhibit similar affinity for
the [3H]DTBZ binding site on VMAT2 and decreased affinity
for nAChRs, thus revealing increased selectivity for VMAT2
(Zheng et al., 2005). In addition, MTD inhibited metham-
phetamine-evoked DA overflow from rat striatal slices (Nick-
ell et al., 2010a). However, MTD exhibited high affinity for
DAT (Miller et al., 2004), which has been associated with the
potential for abuse liability. Furthermore, MTD has limited
solubility, diminishing its potential for development as a
pharmacotherapy for methamphetamine abuse.
To extend the previous work, the current study determined
whether MTD decreases methamphetamine self-administra-
tion in rats. The current SAR also identified analogs based on
the MTD scaffold that potently and selectively inhibit
VMAT2 function and had both low affinity for DAT and
increased water solubility compared with MTD. These ana-
logs were designed as more rigid, conformationally restricted
ABBREVIATIONS: DA, dopamine; DAT, DA transporter; 5-HT, 5-hydroxytryptamine (serotonin); ANOVA, analysis of variance; DOPAC,
dihydroxyphenylacetic acid; DTBZ, dihydrotetrabenazine; GBR 12909, 1-(2-(bis-(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)-
piperazine; MLA, methyllycaconitine; MTD, meso-transdiene; nAChR, nicotinic acetylcholine receptor; PEI, polyethyleneimine; Ro-4-1284,
(2R,3S,11bS)-2-ethyl-3-isobutyl-9,10-dimethoxy-2,2,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinolin-2-ol; SAR, structure-activity rela-
tionship; SERT, serotonin transporter; UKMH-101, (3Z,5E)-3,5-dibenzylidene-1-methylpiperidine; UKMH-102, (3Z,5Z)-3,5-dibenzylidene-1-
methylpiperidine; UKMH-103, (3Z,5E)-1-methyl-3,5-bis((E)-3-phenylallylidene)piperidine; UKMH-104, (3Z,5Z)-1-methyl-3,5-bis((E)-3-
phenylallylidene)piperidine; UKMH-105, (3Z,5E)-3,5-bis(2,4-dichlorobenzylidene)-1-methylpiperidine; UKMH-106, (3Z,5Z)-3,5-bis(2,
4-dichlorobenzylidene)-1-methylpiperidine; UKMH-107, (3Z,5Z)-3,5-bis(4-methoxybenzylidene)-1-methylpiperidine; UKMH-108, (3Z,5Z)-1-
methyl-3,5-bis(4-methylbenzylidene)-piperidine; UKMH-109, (3Z,5Z)-1-methyl-3,5-bis(thiophen-2-ylmethylene)piperidine; UKMH-110,
(3Z,5Z)-1-methyl-3,5-bis(thiophen-3-ylmethylene)piperidine; UKMH-111, (3Z,5Z)-3,5-bis(furan-2-ylmethylene)-1-methylpiperidine; UKMH-
112, (3Z,5Z)-3,5-bis(furan-3-ylmethylene)-1-methylpiperidine; VMAT2, vesicular monoamine transporter; TLC, thin layer chromatography;
HPLC, high-performance liquid chromatography; EC, electrochemical detection.
Fig. 1. Chemical structures of lobeline, MTD, and MTD analogs incorpo-
rating the phenylethylene moiety of MTD into the piperidine ring system
with the addition of various phenyl ring substituents. For clarity of
presentation, compounds are grouped according to structural similarity.
Top, lobeline, MTD, and MTD analogs with no phenyl ring additions.
Middle, MTD analogs with dichloro, methoxy, or methyl additions. Bot-
tom, MTD analogs with heteroaromatic phenyl ring substitutions.
MTD Analogs, Methamphetamine, and VMAT2
analogs of MTD, in which the phenylethylene substituents in
the MTD structure were incorporated into the piperidine ring
system (Fig. 2). This structural change reduces the molecular
weight and the number of rotational carbon bonds from four
in MTD to two in the current analogs. Other changes in-
cluded: 1) altering the geometry of the C5 double bond from
E to Z; 2) lengthening the linker units at C3 and C5 of the
piperidine ring; 3) adding aromatic substituents to the phe-
nyl moieties; and 4) replacing the phenyl rings with het-
eroaromatic rings, such as thiophene or furan. Affinity for
VMAT2 was retained despite these structural alterations,
and importantly, selectivity for VMAT2 was improved. These
novel analogs were evaluated further for their ability to
inhibit methamphetamine-evoked DA release from super-
fused rat striatal slices and constitute new leads in the dis-
covery of novel treatments for methamphetamine abuse.
Materials and Methods
Animals. Male Sprague-Dawley rats (200–250 g; Harlan, India-
napolis, IN) were housed two per cage with ad libitum access to food
and water in the Division of Laboratory Animal Resources at the
University of Kentucky (Lexington, KY). Experimental protocols
involving the animals were in accord with the 1996 National
Institutes of Health Guide for the Care and Use of Laboratory
Animals and approved by the Institutional Animal Care and Use
Committee at the University of Kentucky.
Chemicals. [3H]Nicotine (L-(?)-[N-methyl-3H]; specific activity,
66.9 Ci/mmol), [3H]dopamine ([3H]DA; dihydroxyphenylethylamine,
3,4-[7-3H]; specific activity, 28 Ci/mmol), [3H]5-hydroxytryptamine
([3H]5-HT; hydroxytryptamine creatinine sulfate 5-[1,2-3H(N)];
specific activity, 30 Ci/mmol), and Microscint 20 LSC-cocktail
were purchased from PerkinElmer Life and Analytical Sciences
(Waltham, MA). [3H]Dihydrotetrabenazine ([3H]DTBZ; (?)?-[O-
methyl-3H]dihydrotetrabenazine; specific activity, 20 Ci/mmol) and
[3H]methyllycaconitine ([3H]MLA; ([1?,4(S),6?,14?,16?]-20-ethyl-
nyl)benzoyl]oxy]-methyl]aconitane-7,8-diol; specific activity, 100 Ci/
mmol) were obtained from American Radiolabeled Chemicals, Inc.
(St. Louis, MO). Diazepam and ketamine were purchased from
N.L.S. Animal Health (Pittsburgh, PA). Acetonitrile, ATP-Mg2?,
benzaldehyde, 2,4-dichlorobenzaldehyde, 4-methoxybenzaldehyde,
4-methylbenzaldehyde, furan-2-carbaldehyde, furan-3-carbalde-
hyde, trans-cinnamaldehyde, catechol, DA, dihydroxyphenylacetic
acid (DOPAC), EDTA, EGTA, ethyl acetate, fluoxetine HCl, 1-(2-(bis-
12909), ?-D-glucose, HEPES, hexane, MgSO4, methanol, methylene
chloride, 1-methyl-4-piperidone, pargyline HCl, polyethyleneimine
(PEI), KOH, potassium tartrate, sodium borohydride, NaOH,
Na2SO4, sucrose, silica gel (240–400 mesh), and trifluoroacetic acid
were purchased from Sigma-Aldrich (St. Louis, MO). L-Ascorbic acid
and NaHCO3were purchased from Aldrich Chemical Co. (Milwau-
kee, WI). CaCl2, KCl, K2PO4, MgCl2, NaCl, and NaH2PO4were
purchased from Fisher Scientific Co. (Pittsburgh, PA). Thiophene-
2-carbaldehyde and thiophene-3-carbaldehyde were purchased
from Acros Organics (Fairlawn, NJ). Preparative TLC plates (250
?M silica layer, organic binder, no indicator) were purchased from
Dynamic Adsorbents Inc. (Atlanta, GA). Chloroform-D was pur-
chased from Cambridge Isotope Laboratories, Inc. (Andover, MA).
Complete counting cocktail 3a70B was purchased from Research
Products International Corp. (Mt. Prospect, IL). (2R,3S,11bS)-2-
pyrido[2,1-a]isoquinolin-2-ol (Ro-4-1284) was a generous gift from
Hoffman-LaRoche (Nutley, NJ). MTD was prepared as described
by Zheng et al. (2005) and was used as HCl salt.
General Synthetic Methodology for the UKMH Analogs. A
mixture of 1-methyl-4-piperidone (1.0 eq, 10.2 mmol), the appropri-
ately substituted aromatic aldehyde (2.1 Eq, 21.42 mmol), and po-
tassium hydroxide (2.1 Eq, 21.42 mmol) were stirred in methanol
(20 ml) at ambient temperature for 4 h. The resulting yellow precipitate
was collected by filtration and washed with cold methanol to yield the
89.9–96.4% yield). Without further purification, the crude 3,5-disubsti-
tuted-1-methylpiperidin-4-one product was added to a pre-equilibrated
mixture of sodium borohydride (4 Eq) and trifluoroacetic acid (16 Eq) in
a 1:1 mixture of dichloromethane and acetonitrile. The mixture was
stirred at ambient temperature for 4 to 8 h until TLC and gas chroma-
tography-mass spectroscopy analysis revealed that all of the starting
material was consumed. The reaction mixture was then diluted with
dichloromethane, and 2 M aqueous sodium hydroxide solution was
added drop-wise with stirring to afford a pH of 10. The organic layer
was then separated, dried over anhydrous sodium sulfate, and fil-
tered, and the filtrate evaporated to dryness under vacuum. The
reaction yielded a mixture of mainly the 3Z,5Z- and 3Z,5E-geomet-
rical isomers of the 3,5-disubstituted-1-methylpiperidines, as well as
other minor geometric double-bond combinations; both of these iso-
mers could be separated by silica gel column chromatography or
preparative TLC, using a 10:1 hexane/ethyl acetate solvent mixture.
Using this general procedure, the UKMH series of analogs shown in
Fig. 1 were prepared and fully characterized for structural identity
and purity, as determined by TLC, gas chromatography-mass spec-
converted to HCl salt before analysis.
Methamphetamine Self-Administration. Behavioral experi-
ments were conducted as described previously (Neugebauer et al.,
2007). Operant conditioning chambers (ENV-008; MED Associates,
St. Albans, VT) were enclosed within sound-attenuating compart-
ments (ENV-018M; MED Associates). Each chamber was connected
to a personal computer interface (SG-502; MED Associates), and
chambers were operated using MED-PC software. A 5 ? 4.2-cm
recessed food tray was located on the response panel of each cham-
ber. Two retractable response levers were mounted on either side of
the recessed food tray (7.3 cm above the metal rod floor). A 28-V,
3-cm diameter, white cue light was mounted 6 cm above each re-
Rats were trained briefly to respond on a lever for food reinforce-
ment. Immediately after food training, rats were allowed free access
to food for 3 days. Rats were anesthetized (100 mg/kg ketamine and
5 mg/kg diazepam i.p.), and catheters were implanted into the right
jugular vein, exiting through a dental acrylic head mount affixed to
the skull via jeweler screws. Drug infusions were administered in-
travenously (0.1 ml over 5.9 s) via a syringe pump (PHM-100; MED
Associates) through a water-tight swivel attached to a 10-ml syringe
via catheter tubing, which was attached to the cannulae mounted to
the head of the rat. After a 1-week recovery period from surgery, rats
were trained to press one of two levers for an infusion of metham-
phetamine (0.05 mg/kg/infusion). Each infusion was followed by a
20-s timeout signaled by illumination of both lever lights. The re-
sponse requirement was gradually increased to a terminal fixed ratio
1H NMR and
13C NMR analysis. All analogs were
Fig. 2. Incorporating the phenylethylene moiety of MTD into the piper-
dine ring of the analogs affords a novel more rigid molecule. For all
analogs in the series, the phenylethylene substituents in the MTD struc-
ture (left) were incorporated into the piperidine ring system to afford
analogs (right) with a similar number of carbons between the piperidine
nitrogen and the phenyl rings. This structural change reduces the mo-
lecular weight and the number of rotational carbon bonds (curved arrows)
from four in MTD to two in the MTD analogs, affording a novel, more
conformationally restricted structure.
Horton et al.
5 schedule of reinforcement. Each session was 60 min in duration.
Training continued until responding stabilized across sessions. Sta-
ble responding was defined as less than 20% variability in the num-
ber of infusions earned across three successive sessions, a minimum
of a 2:1 ratio of active (drug) lever responses to inactive (no drug)
lever responses, and at least 10 infusions per session. Once stability
was reached, an acute dose (0, 3.0, 5.6, 10, or 17 mg/kg) of MTD was
administered (subcutaneously) 15 min before the session according
to a within-subject Latin square design. Two maintenance sessions
(i.e., no pretreatment) were included between each test session to
ensure stable responding throughout the experiment.
Food-Maintained Responding. In brief, rats were trained to
respond on one lever (active lever) for food pellet reinforcement
(45-mg pellets; BIO-SERV, Frenchtown, NJ), while responses on the
other lever (inactive lever) had no programmed consequence. Loca-
tions (left or right) of the active and inactive levers were counterbal-
anced across rats. The response requirement was gradually in-
creased, terminating at fixed ratio 5. After lever training, a 20-s
signaled timeout (illumination of both lever lights) was included
after each pellet delivery. Timeout after each pellet delivery was
instituted to be consistent with the methamphetamine self-admin-
istration procedure. Each food-reinforced session lasted 60 min.
Training continued until responding stabilized across sessions. Sta-
ble responding was defined as less than 20% variability in the num-
ber of pellets earned across three successive sessions, and a mini-
mum of a 2:1 ratio of active lever responses to inactive lever
responses. After the stability criteria were reached, an acute dose of
MTD (17 mg/kg) was administered (subcutaneously) 15 min before
the 60-min session. Two maintenance sessions (i.e., no pretreatment)
were included between test sessions to ensure stable responding
throughout the experiment.
[3H]Nicotine and [3H]MLA Binding Assays. Analog-induced
inhibition of [3H]nicotine and [3H]MLA binding was determined
using published methods (Miller et al., 2004). Whole brain, excluding
cortex and cerebellum, was homogenized using a Tekmar polytron
(Tekmar-Dohrmann, Mason, OH) in 20 volumes of ice-cold modified
Krebs’-HEPES buffer containing 2 mM HEPES, 14.4 mM NaCl, 0.15
mM KCl, 0.2 mM CaCl2? ? 2H2O, and 0.1 mM MgSO4? 7H2O, pH 7.5.
Homogenates were centrifuged at 31,000g for 17 min at 4°C (Avanti
J-301 centrifuge; Beckman Coulter, Fullerton, CA). Pellets were
resuspended by sonication (Vibra Cell; Sonics and Materials Inc.,
Danbury, CT) in 20 volumes of Krebs’-HEPES buffer and incubated
at 37°C for 10 min (Reciprocal Shaking Bath model 50; Precision
Scientific, Chicago, IL). Suspensions were centrifuged using the
above conditions. Resulting pellets were resuspended by sonication
in 20 volumes buffer and centrifuged at 31,000g for 17 min at 4°C.
Final pellets were stored in incubation buffer containing 40 mM
HEPES, 288 mM NaCl, 3.0 mM KCl, 4.0 mM CaCl2? 2H2O, and 2.0
mM MgSO4? 7H2O, pH 7.5. Membrane suspensions (100–140 ?g of
protein/100 ?l) were added to duplicate wells containing 50 ?l of
analog (7–9 concentrations, 1 nM-0.1 mM, final concentration), 50 ?l
of buffer, and 50 ?l of [3H]nicotine or [3H]MLA (3 nM; final concen-
tration) for a final volume of 250 ?l and incubated for 1 h at room
temperature. Nonspecific binding was determined in the presence of
10 ?M cytisine or 10 ?M nicotine for the [3H]nicotine and [3H]MLA
assays, respectively. Reactions were terminated by harvesting sam-
ples on Unifilter-96 GF/B filter plates presoaked in 0.5% PEI using a
Packard Filter Mate Harvester (PerkinElmer Life and Analytical
Sciences). Samples were washed three times with 350 ?l of ice-cold
buffer. Filter plates were dried for 60 min at 45°C and bottom-sealed,
and each well was filled with 40 ?l of Microscint 20 cocktail. Bound
radioactivity was determined via liquid scintillation spectrometry
(TopCount NXT scintillation counter; PerkinElmer Life and Analyt-
Synaptosomal [3H]DA and [3H]5-HT Uptake Assays. Analog-
induced inhibition of [3H]DA and [3H]5-HT uptake into rat striatal
and hippocampal synaptosomes, respectively, was determined using
modifications of a previously described method (Teng et al., 1997).
Brain regions were homogenized in 20 ml of ice-cold 0.32 M sucrose
solution containing 5 mM NaHCO3, pH 7.4, with 16 up-and-down
strokes of a Teflon pestle homogenizer (clearance ? 0.005 inch).
Homogenates were centrifuged at 2000g for 10 min at 4°C, and
resulting supernatants were centrifuged at 20,000g for 17 min at
4°C. Pellets were resuspended in 1.5 ml of Krebs’ buffer, containing
125 mM NaCl, 5 mM KCl, 1.5 mM MgSO4, 1.25 mM CaCl2, 1.5 mM
KH2PO4, 10 mM ?-D-glucose, 25 mM HEPES, 0.1 mM EDTA, with
0.1 mM pargyline and 0.1 mM ascorbic acid saturated with 95% O2/
5% CO2, pH 7.4. Synaptosomal suspensions (20 ?g of protein/50 ?l)
were added to duplicate tubes containing 50 ?l of analog (7–9 con-
centrations, 0.1 nM-1 mM, final concentration) and 350 ?l of buffer
and incubated at 34°C for 5 min in a total volume of 450 ?l. Samples
were placed on ice and 50 ?l of [3H]DA or [3H]5-HT (10 nM; final
concentration) was added to each tube for a final volume of 500 ?l.
Reactions proceeded for 10 min at 34°C and were terminated by the
addition of 3 ml of ice-cold Krebs’ buffer. Nonspecific [3H]DA and
[3H]5-HT uptake were determined in the presence of 10 ?M GBR
12909 and 10 ?M fluoxetine, respectively. Samples were rapidly
filtered through Whatman GF/B filters using a cell harvester (MP-
43RS; Brandel Inc., Gaithersburg, MD). Filters were washed three
times with 4 ml of ice-cold Krebs’ buffer containing catechol (1 ?M).
Complete counting cocktail was added to the filters and radioactivity
was determined by liquid scintillation spectrometry (B1600 TR scin-
tillation counter; PerkinElmer Life and Analytical Sciences).
[3H]DTBZ Vesicular Binding Assays. Analog-induced inhibi-
tion of [3H]DTBZ binding, a high-affinity ligand for VMAT2, was
determined using modifications of a previously published method
(Teng et al., 1998). Rat whole brain (excluding cerebellum) was
homogenized in 20 ml of ice-cold 0.32 M sucrose solution with 10
up-and-down strokes of a Teflon pestle homogenizer (clearance ?
0.008 inch). Homogenates were centrifuged at 1000g for 12 min at
4°C, and the resulting supernatants were centrifuged at 22,000g for
10 min at 4°C. Resulting pellets were osmotically shocked by incu-
bation in 18 ml of cold water for 5 min. Osmolarity was restored by
adding 2 ml of 25 mM HEPES and 100 mM potassium tartrate
solution. Samples were centrifuged (20,000g for 20 min at 4°C), and
then 1 mM MgSO4solution was added to the supernatants. Samples
were centrifuged at 100,000g for 45 min at 4°C. Pellets were resus-
pended in cold assay buffer, containing 25 mM HEPES, 100 mM
potassium tartrate, 5 mM MgSO4, 0.1 mM EDTA, and 0.05 mM
EGTA, pH 7.5. Assays were performed in duplicate in 96-well plates.
Vesicular suspensions (15 ?g of protein/100 ?l) were added to wells
containing 50 ?l of analog (7–9 concentrations, 0.01 nM-0.1 mM,
final concentration), 50 ?l of buffer, and 50 ?l of [3H]DTBZ (3 nM;
final concentration) for a final volume of 250 ?l and incubated for 1 h
at room temperature. Nonspecific uptake was determined in the
presence of 50 ?l of 20 ?M Ro-4-1284. Reactions were terminated by
filtration onto Unifilter-96 GF/B filter plates (presoaked in 0.5%
PEI). Filters were washed three times with 350 ?l of ice-cold buffer
containing 25 mM HEPES, 100 mM potassium-tartrate, 5 mM
MgSO4, and 10 mM NaCl, pH 7.5. Filter plates were dried and
bottom-sealed and each well was filled with 40 ?l of scintillation
cocktail (MicroScint 20; PerkinElmer Life and Analytical Sciences).
Radioactivity on the filters was determined by liquid scintillation
Vesicular [3H]DA Uptake Assay. Analog-induced inhibition of
[3H]DA uptake into rat striatal vesicles was determined using mod-
ifications of a previously published method (Teng et al., 1997). Pre-
vious reports from our laboratory showed that this vesicle prepara-
tion contains ?1% contaminating membrane fragments (Teng et al.,
1997). Striata were homogenized in 14 ml of ice-cold 0.32 M sucrose
solution containing 5 mM NaHCO3, pH 7.4, with 10 up-and-down
strokes of a Teflon pestle homogenizer (clearance ? 0.008 inch).
Homogenates were centrifuged at 2000g for 10 min at 4°C, and the
resulting supernatants were centrifuged at 10,000g for 30 min at
4°C. Pellets were resuspended in 2.0 ml of 0.32 M sucrose, trans-
ferred to tubes containing 7 ml of MilliQ water, and homogenized
MTD Analogs, Methamphetamine, and VMAT2
with five up-and-down strokes. Homogenates were transferred to
tubes containing 900 ?l of 0.25 M HEPES and 900 ?l of 1.0 M
potassium tartrate solution and centrifuged at 20,000g for 20 min at
4°C. The resulting supernatants were centrifuged at 55,000g for 60
min at 4°C. Subsequently, 100 ?l of 1 mM MgSO4, 100 ?l of 0.25 M
HEPES, and 100 ?l of 1.0 M potassium tartrate were added to the
supernatant and centrifuged at 100,000g for 45 min at 4°C. Final
pellets were resuspended in assay buffer containing 25 mM HEPES,
100 mM potassium tartrate, 50 ?M EGTA, 100 ?M EDTA, 1.7 mM
ascorbic acid, and 2 mM ATP-Mg2?, pH 7.4. Vesicular suspensions
(10 ?g of protein/100 ?l) were added to duplicate tubes containing 50
?l of analog (7–9 concentrations, 1 nM-0.1 mM, final concentration),
300 ?l of buffer, and 50 ?l of [3H]DA (0.1 ?M; final concentration) for
a final volume of 500 ?l and incubated for 8 min at 34°C. Nonspecific
[3H]DA uptake was determined in the presence of 10 ?M Ro-4-1284.
Samples were filtered rapidly through Whatman GF/B filters using
the cell harvester and washed three times with assay buffer contain-
ing 2 mM MgSO4in the absence of ATP. Radioactivity retained by
the filters was determined as described previously.
Kinetics of Vesicular [3H]DA Uptake. Vesicle suspensions
were prepared as described above; striata were pooled from two rats.
Vesicular suspensions (20 ?g of protein/50 ?l) were added to dupli-
cate tubes containing 25 ?l of analog (final concentration approxi-
mating the Ki), 150 ?l of buffer, and 25 ?l of [3H]DA (1 nM-5 ?M;
final concentration) for a final volume of 250 ?l and incubated for 8
min at 34°C. Nonspecific [3H]DA uptake was determined in samples
containing 10 ?M Ro-4-1284. Samples were processed as described
Endogenous DA Release Assay. Rat coronal striatal slices (0.5
mm thick) were prepared and incubated in Krebs’ buffer containing
118 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 1.0 mM NaH2PO4, 1.3
mM CaCl2, 11.1 mM ?-D-glucose, 25 mM NaHCO3, 0.11 mM
L-ascorbic acid, and 0.004 mM EDTA, pH 7.4, saturated with 95%
O2/5% CO2at 34°C in a metabolic shaker for 60 min (Teng et al.,
1997). Each slice was transferred to a glass superfusion chamber and
superfused at 1 ml/min for 60 min with Krebs’ buffer before sample
collection. Two basal samples (1 ml) were collected at 5- and 10-min
time points. Each slice was superfused for 30 min in the absence or
presence of a single concentration of analog (0.1–10 ?M) to deter-
mine analog-evoked DA and DOPAC overflow and remained in the
buffer until the end of the experiment. Methamphetamine (5 ?M)
was added to the buffer after 30 min of superfusion, and slices were
superfused for 15 min, followed by 20 min of superfusion in the
absence of methamphetamine. In each experiment, a striatal slice
was superfused for 90 min in the absence of both analog and meth-
amphetamine, serving as the buffer control condition. In each exper-
iment, duplicate slices were superfused with methamphetamine in
the absence of analog, serving as the methamphetamine control
condition. The concentration of methamphetamine was selected
based on pilot concentration-response data showing a reliable re-
sponse of sufficient magnitude to allow evaluation of analog-induced
inhibition. Each superfusate sample (1 ml) was collected into tubes
containing 100 ?l of 0.1 M perchloric acid. Before HPLC-EC analysis,
ascorbate oxidase (20 ?l, 168 U/mg reconstituted to 81 U/ml) was
added to 500 ?l of each sample and vortexed for 30 s, and 100 ?l of
the resulting solution was injected onto the HPLC-EC.
HPLC-EC consisted of a pump (model 126; Beckman Coulter) and
autosampler (model 508; Beckman Coulter), an ODS Ultrasphere
C18 reverse-phase 80 ? 4.6 mm, 3-?m column, and a Coulometric-II
detector with guard cell (model 5020) maintained at ? 0.60 V and
analytical cell (model 5011) maintained at potentials E1 ? ?0.05 V
and E2 ? ?0.32 V (ESA Inc., Chelmsford, MA). HPLC mobile phase
(flow rate, 1.5 ml/min) was 0.07 M citrate/0.1 M acetate buffer, pH 4,
containing 175 mg/l octylsulfonic acid sodium salt, 650 mg/l NaCl.
and 7% methanol. Separations were performed at room temperature,
and 5 to 6 min was required to process each sample. Retention times
of DA or DOPAC standards were used to identify respective peaks.
Peak heights were used to quantify the detected amounts of analyte
based on standard curves. Detection limit for DA and DOPAC was 1
to 2 pg/100 ?l.
Data Analysis. For the behavioral experiments, one-way ANOVA
with dose as a within-subject factor was used to determine whether
MTD altered methamphetamine self-administration. Dunnett’s post
hoc test were used to compare each MTD dose to the saline control.
A single paired-sample t test was used to determine the effects of
MTD on food-maintained behavior.
For the neurochemical experiments, specific [3H]nicotine, [3H]MLA,
and [3H]DTBZ binding and specific [3H]DA and [3H]5-HT uptake were
determined by subtracting the nonspecific binding or uptake from the
total binding or uptake. Analog concentrations producing 50% inhibi-
tion of specific binding or uptake (IC50values) were determined from
concentration effect curves via an iterative curve-fitting program
(Prism 5.0; GraphPad Software Inc., San Diego, CA). Inhibition con-
stants (Kivalues) were determined using the Cheng-Prusoff equation
(Cheng and Prusoff, 1973). For kinetic analyses, Kmand Vmaxwere
determined using one-site binding curves. Paired two-tailedt tests were
performed on the arithmetic Vmaxand the log Kmvalues to determine
significant differences between analog and control conditions. Pearson’s
correlation analysis determined the relationship between affinity for
the [3H]DTBZ binding site and vesicular [3H]DA uptake.
For endogenous neurotransmitter release assays, fractional release
was defined as the DA or DOPAC concentration in each sample divided
by the slice weight. Basal DA or DOPAC outflow was calculated as the
average fractional release of the two basal samples collected 10 min
before addition of analog to the buffer. Analog-evoked DA or DOPAC
overflow was calculated as the average fractional release during the
Fig. 3. MTD dose-dependently decreases methamphetamine self-admin-
istration, without altering food-maintained responding. Top, dose-related
effect of acute MTD on methamphetamine (METH) self-administration.
Bottom, effect of the high dose of MTD (17.0 mg/kg) on food-maintained
responding. Data are expressed as mean ? S.E.M. for number of meth-
amphetamine infusions (0.05 mg/kg/infusion) or number of pellets earned
during 60-min sessions (n ? 5–6). ??, p ? 0.01 compared with control.
Horton et al.
30-min period of analog exposure before methamphetamine addition to
the buffer. Analog-evoked DA or DOPAC overflow was analyzed by
one-way repeated-measures ANOVA. Time course for analog-induced
inhibition of methamphetamine-evoked fractional DA or DOPAC re-
lease was analyzed by two-way ANOVA with concentration and time as
repeated-measures factors. If a concentration ? time interaction was
found, one-way ANOVAs were performed at each time point at which
methamphetamine-evoked DA release above basal outflow. When ap-
propriate, one-way ANOVAs were followed by Dunnett’s post hoc test to
determine concentrations of analog that decreased methamphetamine-
evoked DA fractional release. Furthermore, one-way ANOVA was per-
formed on the peak response of methamphetamine-evoked fractional
release at each analog concentration. The log IC50value was generated
using an iterative nonlinear least-squares curve-fitting program (Prism
version 5.0). Statistical significance was defined as p ? 0.05.
MTD Decreases Methamphetamine Self-Administra-
tion Without Altering Food-Maintained Responding. The
effect of MTD on methamphetamine self-administration is il-
lustrated in Fig. 3, top. One-way ANOVA revealed a dose-
related effect of MTD on the number of methamphetamine
infusions earned (F4,16? 4.86; p ? 0.01). Dunnett’s test re-
vealed that the high dose of MTD (17 mg/kg) decreased the
number of methamphetamine infusions earned compared with
control. Tolerance developed to the ability of MTD to decrease
methamphetamine self-administration on the second day of
treatment (data not shown). The effect of the acute high dose of
Fig. 3, bottom. MTD did not decrease responding for food (p ?
0.414). Thus, the high dose of MTD specifically decreased meth-
amphetamine self-administration; however, tolerance devel-
oped to this effect.
MTD Analogs Do Not Inhibit [3H]Nicotine and [3H]MLA
Binding. Concentration-response curves and Kivalues for lo-
beline, MTD, and the series of MTD analogs to inhibit [3H]ni-
cotine and [3H]MLA binding to whole brain membranes, com-
pared with nicotine (positive control), are provided in
Supplemental Fig. 1 (top and bottom, respectively) and Table 1.
[3H]MLA binding sites, respectively, consistent with previous
reports (Flammia et al., 1999). Kivalues for lobeline were 4 nM
and 6.26 ?M at the [3H]nicotine and [3H]MLA binding sites,
respectively, also consistent with previous reports (Zheng et al.,
2005). Kivalues for MTD were ?100 ?M at both [3H]nicotine
and [3H]MLA binding sites, as observed previously (Miller et
al., 2004). None of the MTD analogs in this series inhibited
[3H]nicotine or [3H]MLA binding.
MTD Analogs Inhibit Synaptosomal [3H]DA Uptake.
Concentration-response curves and Kivalues for lobeline, MTD,
and the series of MTD analogs to inhibit [3H]DA uptake into
trol), are provided in Fig. 4 and Table 1. The Kivalue for GBR
12909 to inhibit [3H]DA uptake was 0.97 nM, consistent with
previous reports (Reith et al., 1994). The Kivalue for lobeline to
inhibit [3H]DA uptake was 28.2 ?M, whereas the defunctional-
ized unsaturated compound MTD exhibited a 200-fold higher
potency (Ki? 100 nM) compared with lobeline, in agreement
with previous observations (Miller et al., 2004). MTD analogs in
the current series exhibited 50- to 1000-fold lower potency (Ki?
5 ?M) than MTD at DAT. It is noteworthy that the 2,4-dichlo-
rophenyl analogs, (3Z,5E)-3,5-bis(2,4-dichlorobenzylidene)-1-
methylpiperidine (UKMH-105) and (3Z,5Z)-3,5-bis(2,4-dichlo-
MTD. Thus, this series of MTD analogs exhibited lower affini-
ties for DAT compared with the parent compound.
MTD Analogs Inhibit Synaptosomal [3H]5-HT Uptake.
Concentration-response curves and Kivalues for lobeline, MTD,
and the series of MTD analogs to inhibit [3H]5-HT uptake into
hippocampal synaptosomes, compared with fluoxetine (positive
control), are provided in Fig. 5 and Table 1. The Kivalue for
fluoxetine to inhibit [3H]5-HT uptake was 6.5 nM, consistent
with previous reports (Owens et al., 2001). The Kivalue for
exhibited 6-fold higher potency (Ki? 7 ?M) compared with
lobeline, in agreement with previous observations (Miller et al.,
Affinity values (Ki) of MTD analogs, lobeline, MTD, and standard compounds for nicotinic receptors, DAT, SERT, and VMAT2 binding
DAT ?3H?DA Uptake
0.00097 ? 0.0001a
28.2 ? 6.73
0.10 ? 0.01
11.5 ? 1.90
25.1 ? 2.93
16.2 ? 1.20
5.25 ? 0.46
6.27 ? 0.60
6.90 ? 1.10
68.2 ? 6.93
39.0 ? 16.3
58.1 ? 18.7
5.50 ? 0.26
SERT ?3H?5-HT Uptake
VMAT2 ?3H?DTBZ Binding
VMAT2 ?3H?DA Uptake
0.003 ? 0.0002a
0.004 ? 0.0001
0.37 ? 0.08a
6.26 ? 1.30
0.0065 ? 0.0001a
46.8 ? 3.70
7.00 ? 1.30
0.71 ? 0.09
1.37 ? 0.09
2.10 ? 0.51
2.67 ? 0.51
18.3 ? 7.50
20.7 ? 4.90
0.51 ? 0.05
0.61 ? 0.08
16.3 ? 4.10
13.4 ? 4.10
16.1 ? 2.91
0.71 ? 0.19
0.028 ? 0.003a
2.04 ? 0.26b
9.88 ? 2.22b
31.8 ? 5.84
12.3 ? 4.70
20.3 ? 3.73
15.0 ? 5.22
4.60 ? 1.70
41.3 ? 14.3
7.27 ? 2.28
3.42 ? 0.26
91.3 ? 26.2
10.4 ? 2.62
32.6 ? 6.79
15.5 ? 1.61
0.018 ? 0.002a
1.27 ? 0.46
0.46 ? 0.11
0.88 ? 0.19
0.22 ? 0.05
0.79 ? 0.18
0.88 ? 0.26
0.22 ? 0.01
0.32 ? 0.12
1.03 ? 0.19
0.33 ? 0.08
2.27 ? 1.13
0.36 ? 0.12
3.82 ? 1.99
0.58 ? 0.08
N.D., not determined. UKMH-110, (3Z,5Z)-1-methyl-3,5-bis(thiophen-3-ylmethylene)piperidine; UKMH-111, (3Z,5Z)-3,5-bis(furan-2-ylmethylene)-1-methylpiperidine.
an ? 3–4 rats.
bData taken from Zheng et al., 2005.
cData taken from Miller et al., 2001.
MTD Analogs, Methamphetamine, and VMAT2
2004). The majority of the MTD analogs had Kivalues not
different from MTD; of note, the 2,4-dichlorophenyl analogs,
UKMH-105 and UKMH-106, exhibited low potency at SERT
(Ki? 18.3 and 20.7 ?M, respectively). Exceptions include
(3Z,5E)-3,5-dibenzylidene-1-methylpiperidine (UKMH-101) (no
phenyl substituents), (3Z,5Z)-3,5-bis(4-methoxybenzylidene)-1-
methylpiperidine (UKMH-107) (a 4-methoxyphenyl analog),
(UKMH-108) (a 4-methylphenyl analog), and (3Z,5Z)-3,5-
(a 3-furanyl analog), which exhibited 10-fold higher potency
at SERT compared with MTD.
MTD Analogs Inhibit [3H]DTBZ Binding at VMAT2.
Concentration-response curves and Kivalues for lobeline, MTD,
and the series of MTD analogs to inhibit [3H]DTBZ binding to
whole brain membranes, compared with Ro-4-1284 (positive con-
to inhibit [3H]DTBZ binding was 28 nM, consistent with a previ-
ous report (Cesura et al., 1990). The Kivalue for lobeline to inhibit
[3H]DTBZ binding was 2.04 ?M, whereas MTD exhibited a 5-fold
lower potency (Ki? 9.88 nM) compared with lobeline, consistent
with previous observations (Zheng et al., 2005). The majority of
yl-3,5-bis(thiophen-2-ylmethylene)piperidine (UKMH-109) (2-
[3H]DTBZ binding site compared with MTD. It is noteworthy
that UKMH-105 and UKMH-106, the 2,4-dichlorophenyl dou-
ble-bond isomers, exhibited geometrically specific inhibition of
[3H]DTBZ binding (Ki? 4.60 and 41.3 ?M, respectively).
MTD Analogs Inhibit [3H]DA Uptake by VMAT2. Con-
centration-response curves and Kivalues for lobeline, MTD,
and the series of MTD analogs to inhibit [3H]DA uptake into
striatal vesicles, compared with Ro-4-1284 (positive control),
Fig. 4. Structural modifications to MTD afford analogs with decreased
affinity for DAT. Analogs are grouped according to structural similarity of
the aromatic rings. Top, lobeline, MTD, and MTD analogs with no aro-
matic ring substituents. Middle, MTD analogs with dichloro, methoxy, or
methyl aromatic substituents. Bottom, MTD analogs containing het-
eroaromatic rings. MTD is repeated in all three panels for purpose of
comparison. Nonspecific [3H]DA uptake was determined in the presence
of 10 ?M GBR 12909. Control (CON) represents specific [3H]DA uptake in
the absence of analog (35.0 ? 1.55 pmol/mg/min). Legends provide ana-
logs in order from highest to lowest affinity. n ? 4 rats/analog.
Fig. 5. MTD analogs inhibit [3H]5-HT uptake into rat hippocampal syn-
aptosomes. Analogs are grouped according to structural similarity of the
aromatic rings. Top, lobeline, MTD, and MTD analogs with no aromatic
ring substituents. Middle, MTD analogs with dichloro, methoxy, or
methyl aromatic substituents. Bottom, MTD analogs containing het-
eroaromatic rings. MTD is repeated in all three panels for purpose of
comparison. Nonspecific [3H]5-HT uptake was determined in the pres-
ence of 10 ?M fluoxetine. Control (CON) represents specific [3H]5-HT
uptake in the absence of analog (1.67 ? 0.09 pmol/mg/min). Legends
provide compounds in order from highest to lowest affinity. n ? 4 rats/
Horton et al.
are provided in Fig. 7 and Table 1. The Kivalue for Ro-4-1284 to
inhibit [3H]DA uptake was 18 nM, consistent with a previous
report (Nickell et al., 2010b). The Kivalue for lobeline to inhibit
[3H]DA uptake by VMAT2 was 1.27 ?M, which was not differ-
ent from that for MTD (Ki? 0.46 ?M), consistent with previous
observations (Nickell et al., 2010a). The majority of the analogs
in this series were equipotent with MTD inhibiting [3H]DA
uptake at VMAT2 (Table 1). It is noteworthy that the 2,4-
dichlorophenyl isomers, UKMH-105 and UKMH-106, were two
of the most potent analogs in the series, with Kivalues of 0.22
and 0.32 ?M, respectively.
Figure 8 illustrates Kivalues for inhibition of vesicular
[3H]DA uptake as a function of Kifor inhibition of [3H]DTBZ
binding for lobeline, MTD, and the series of MTD analogs.
Correlation analysis revealed no relationship between these
parameters probing VMAT2 (Pearson’s correlation coefficient
r ? 0.42; p ? 0.13).
[3H]DA Uptake at VMAT2. UKMH-105 and UKMH-106 were
20- to 450-fold selective for VMAT2 over DAT, SERT, and
?4?2* and ?7* nAChRs (* indicates putative nAChR subtype
assignment). UKMH-105 and UKMH-106 had 10- to 100-fold
higher affinity in the VMAT2 functional assay compared with
the VMAT2 binding assay. To further evaluate these two ana-
logs, kinetic analyses of [3H]DA uptake at VMAT2 were con-
ducted to determine the mechanism of inhibition, i.e., compet-
itive or noncompetitive, compared with parent compounds
(MTD and lobeline). Kinetic assays revealed an increased Km
value and no change in Vmaxfor each compound (Fig. 9) com-
pared with control, indicating a competitive mechanism of
UKMH-106 Inhibits Methamphetamine-Evoked En-
dogenous DA Release, Whereas UKMH-105 Does Not.
The ability of UKMH-105 and UKMH-106 to evoke DA release
and UKMH-106 CompetitivelyInhibit
Fig. 6. MTD analogs inhibit [3H]DTBZ binding to vesicle membranes
from rat whole brain preparations. Analogs are grouped according to
structural similarity of the aromatic rings. Top, lobeline, MTD, and MTD
analogs with no aromatic ring substituents. Middle, MTD analogs with
dichloro, methoxy, or methyl aromatic substituents. Bottom, MTD ana-
logs containing heteroaromatic rings. MTD is repeated in all three panels
for purpose of comparison. Nonspecific [3H]DTBZ binding was deter-
mined in the presence of 10 ?M Ro-4-1284. Control (CON) represents
specific [3H]DTBZ binding in the absence of analog (5.01 ? 0.10 pmol/mg
protein). Analogs are arranged in order from greatest potency to least
potency. n ? 4 rats/analog.
Fig. 7. MTD analogs inhibit [3H]DA uptake into rat striatal vesicles.
Analogs are grouped according to structural similarity of the aromatic
rings. Top, lobeline, MTD, and MTD analogs with no aromatic ring
substituents. Middle, MTD analogs with dichloro, methoxy, or methyl
aromatic substituents. Bottom, MTD analogs containing heteroaromatic
rings. MTD is repeated in all three panels for purpose of comparison.
Nonspecific [3H]DA uptake was determined in the presence of 10 ?M
Ro-4-1284. Control (CON) represents specific vesicular [3H]DA uptake in
the absence of analog (29.3 ? 1.38 pmol/mg/min). Legends provide com-
pounds in order from highest to lowest affinity. n ? 4 rats/analog.
MTD Analogs, Methamphetamine, and VMAT2
from superfused striatal slices is illustrated in Figs. 10 and 11.
Analysis of the effect of UKMH-105 on DA release before the
addition of methamphetamine to the buffer (20–40 min of sam-
ple collection) showed no main effects of concentration (F5,29?
0.47; p ? 0.05) and time (F4,29? 1.01; p ? 0.05) and no con-
centration ? time interaction (F20,29? 0.67; p ? 0.05). Thus,
UKMH-105 alone did not evoke DA release. Likewise, UKMH-
106 did not alter DA release [no main effect of concentration
(F4,43? 0.12; p ? 0.05) and showed no time ? concentration
interaction (F16,43? 1.57; p ? 0.05)]. A main effect of time was
found (F4,43? 6.78, p ? 0.05), revealing that fractional release
increased slightly across the 20-min exposure period in both the
absence and presence of UKMH-106. Both UKMH-105 and
UKMH-106 also had no effect on DOPAC fractional release
across this time period (data not shown).
The ability of UKMH-105 and UKMH-106 to decrease
methamphetamine-evoked DA release is illustrated in Figs.
10 and 11. A two-way repeated measures ANOVA on frac-
tional DA release during exposure to UKMH-105 and meth-
amphetamine revealed no main effect of concentration (F5,29?
0.65; p ? 0.05) and no concentration ? time interaction
(F25,29? 0.45; p ? 0.05); however, a main effect of time (F5,29?
15.4, p ? 0.0001) was observed, which reflects the increase in
fractional release evoked by methamphetamine in the ab-
sence and presence of UKMH-105. Similarly, only a main
effect of time was found for DOPAC superfusate concentra-
tions; although in the absence and presence of UKMH-105,
DOPAC fractional release was decreased in response to
methamphetamine (data not shown). Thus, UKMH-105 did
not alter the effect of methamphetamine on DA or DOPAC
DA release evoked by methamphetamine (Fig. 11). Two-way
repeated-measures ANOVA on fractional DA release during
exposure to UKMH-106 and methamphetamine revealed a
main effect of concentration (F4,43? 7.61; p ? 0.0001) and time
(F5,43? 23.0; p ? 0.0001) and a concentration ? time interac-
tion (F20,43? 1.68; p ? 0.05). Post hoc analysis revealed that
UKMH-106 (1.0 and 3.0 ?M) decreased methamphetamine-
to 60 min, respectively. The concentration response for UKMH-
106 to inhibit methamphetamine-evoked DA release at peak
response is illustrated in Fig. 11. IC50and Imaxvalues were
0.38 ? 0.13 ?M and 50.2 ? 15.5%, respectively. One-way
ANOVA on peak response data revealed a concentration-depen-
dent effect of UKMH-106 (F4,43? 3.11; p ? 0.05). Post hoc
Fig. 8. Inhibition of [3H]DTBZ binding does not predict inhibition of
[3H]DA uptake at VMAT2. Data presented are Kivalues from analog-
induced inhibition of [3H]DTBZ binding and [3H]DA uptake at VMAT2
(Figs. 6 and 7, respectively). Pearson’s correlation analysis of these data
revealed a lack of correlation (Pearson’s r ? 0.42; p ? 0.13) between the
ability of analogs to inhibit [3H]DTBZ binding and [3H]DA uptake at
Fig. 9. Lobeline, MTD, and MTD analogs competitively inhibit [3H]DA
uptake into vesicles prepared from rat striatum. Concentrations of lobe-
line (0.25 ?M), MTD (0.23 ?M), UKMH-105 (0.11 ?M), and UKMH-106
(0.16 ?M) approximated the Kivalues for inhibiting [3H]DA uptake into
isolated synaptic vesicles obtained from the data shown in Fig. 7. Km(top)
and Vmax(bottom) values are mean ? S.E.M. ?, p ? 0.05 different from
control; ??, p ? 0.01 different from control. n ? 4–7 rats/analog).
Fig. 10. UKMH-105 does not inhibit methamphetamine-evoked endoge-
nous DA release from striatal slices. Fractional DA release represents the
amount of DA in each 5-min sample. Slices were superfused with UKMH-
105 after 10-min collection of basal samples, as indicated by the arrow,
and analog remained in the buffer until the end of the experiment.
Methamphetamine (METH; 5 ?M) was added to the buffer for 15 min as
indicated by the horizontal bar. Fractional release data are expressed as
mean ? S.E.M. pg/ml/mg of the slice weight. n ? 5 rats.
Horton et al.
analysis revealed that 3 ?M UKMH-106 inhibited the DA peak
response. In contrast to the ability of UKMH-106 to decrease
methamphetamine-evoked fractional DA release, fractional
DOPAC release was not altered (data not shown).
In the current study, MTD was shown to decrease metham-
phetamine self-administration specifically, but only at the high-
est dose evaluated, and tolerance developed rapidly to this
effect. Taking into account this encouraging finding, but tem-
pered by the limitations associated with the development of
tolerance, modifications to the MTD molecule were evaluated in
search of preclinical candidates for the treatment of metham-
phetamine abuse. SAR identified several conformationally re-
stricted MTD analogs with high affinity and selectivity for
VMAT2. Structural modifications included lengthening the
linker units, introduction of 4-methoxy, 4-methyl, or 2,4-di-
chloro substituents into the phenyl rings, or replacement of the
phenyl rings with thiophene or furan rings. Effects of altering
the geometry of the double bond at the C5-position of the pip-
eridine ring were evaluated in analogs with either a lengthened
linker unit or an aromatic 2,4-dichloro substituted phenyl ring.
Affinity for VMAT2 was retained, and increases in selectivity
for VMAT2 over DAT were found. The most selective analogs
inhibited methamphetamine-evoked DA release in a geometri-
cally specific manner.
Conformational restriction in combination with both E and
Z geometries at the C5 position of the piperidine ring
[UKMH-101 and (3Z,5Z)-3,5-dibenzylidene-1-methylpiperi-
dine (UKMH-102), respectively] did not alter affinity for
VMAT2 binding and uptake sites. Lengthening the linker
units, regardless of E or Z geometry [(3Z,5E)-1-methyl-3,5-
bis((E)-3-phenylallylidene)piperidine (UKMH-103) and (3Z,
104), respectively], or adding aromatic 4-methoxy or 4-
methyl substituents (UKMH-107 and UKMH-108, respec-
tively), did not alter VMAT2 binding and function. Adding
aromatic electron-withdrawing 2,4-dichloro groups in com-
bination with E or Z geometries at the C5-postion on the
piperidine ring (UKMH-105 and UKMH-106, respectively)
afforded equipotent inhibition of uptake compared with
MTD. In kinetic analyses, UKMH-105 and UKMH-106 in-
creased Kmand did not alter Vmax, indicating competitive
inhibition of DA uptake. Although no differences in affinity
for VMAT2 uptake sites were observed, geometrically spe-
cific inhibition of [3H]DTBZ binding was observed. Specif-
ically, UKMH-106 (3Z,5Z geometry) had 10-fold lower af-
finity than UKMH-105 (3Z,5E geometry) at the [3H]DTBZ
binding site. In contrast, double-bond geometry was not a
contributing factor to affinity for VMAT2 binding or up-
take in analogs (UKMH-101 and UKMH-102) with no phe-
nyl ring substituents or analogs (UKMH-103 and UKMH-104)
with lengthened linker units and no phenyl ring substituents.
Although E geometry was better tolerated than Z geometry at
the VMAT2 binding site, double-bond geometry was not a factor
for affinity at the VMAT2 uptake site.
Substitution of the phenyl rings with thiophene or furan
moieties afforded analogs with equipotent or 10-fold lower
affinity for VMAT2 binding and uptake sites, compared with
MTD. Substitution position on the heteroaromatic ring was a
factor influencing affinity. Specifically, 3-substituted analogs
were equipotent at VMAT2 binding and uptake sites com-
pared with MTD, whereas 2-substituted analogs exhibited
10-fold lower potency. These results suggest that VMAT2 can
accommodate analogs in which furanyl and thiophenyl rings
have been substituted for phenyl rings, with the 3-positional
isomer better tolerated than the 2-positional isomer.
The current results provide examples of structural modifi-
cations that dissociate affinity for the VMAT2 binding site
from that for the VMAT2 substrate site and support previous
observations showing a lack of correlation between affinities
for these sites (Nickell et al., 2010b). The best examples from
the current series of analogs are the 2,4-dichlorophenyl an-
alogs (UKMH-105 and UKMH-106), which were equipotent
at the VMAT2 uptake site, yet exhibited a 10-fold difference
in affinity at the VMAT2 binding site. Thus, these findings
support an interaction at two alternate sites on VMAT2
associated with distinct pharmacophores.
One goal of this study was to discover MTD analogs with
greater selectivity for VMAT2 over DAT. MTD had low affin-
Fig. 11. In a concentration-dependent manner, UKMH-106 inhibits
methamphetamine-evoked DA release in striatal slices. Top, fractional
DA release represents the amount of DA in each 5-min sample. Slices
were superfused with UKMH-106 after 10-min collection of basal sam-
ples, as indicated by the arrow, and analog remained in the buffer until
the end of the experiment. Methamphetamine (METH; 5 ?M) was added
to the buffer for 15 min as indicated by the horizontal bar. Bottom,
concentration-response curve was derived from peak response data for
each concentration of UKMH-106. Fractional release and peak response
data are expressed as mean ? S.E.M. pg/ml/mg of the slice weight. For
fractional release, ?, p ? 0.05 different from methamphetamine alone.
For peak response, ?, p ? 0.05 different from peak response of metham-
phetamine alone (CON). n ? 8 rats.
MTD Analogs, Methamphetamine, and VMAT2
ity (Ki?100 ?M) at ?4?2* and ?7* nAChRs and inhibited DA
uptake by DAT (Ki? 500 nM) and 5-HT uptake by SERT
(Ki? 8.9 ?M) (Miller et al., 2004). Psychostimulant-induced
inhibition of DAT function resulted in increases in extracel-
lular DA, leading to reward and abuse (Ritz et al., 1987;
Williams and Galli, 2006). Affinity of MTD for DAT (Ki? 100
nM; current study) is similar to that for cocaine and methyl-
phenidate (Ki? 300 and 100 nM, respectively) (Han and Gu,
2006), suggesting that MTD may have abuse liability. Reduc-
ing affinity for DAT is imperative to avoiding abuse liability.
Analogs in the current series had reduced affinity (50- to
1000-fold) at DAT compared with MTD. Substitution of the
phenyl rings with 3-thiophenyl and 3-furanyl rings resulted
in the greatest decreases in DAT affinity. Thus, the current
analogs have increased selectivity for VMAT2 over DAT,
compared with MTD, and would be predicted to have reduced
Because MTD had moderate affinity for SERT (Miller et
al., 2004), affinity of the MTD analogs for SERT also was
evaluated. Introduction of aromatic 4-methoxy or 4-methyl
substituents into the phenyl rings of MTD resulted in a 5- to
10-fold increased affinity for SERT compared with MTD. The
remaining structural changes to MTD did not alter affinity at
SERT compared with MTD.
Because the 2,4-dichlorophenyl analogs, UKMH-105 and
UKMH-106, exhibited high affinity and selectivity for inhib-
iting VMAT2 function, these compounds were evaluated for
their ability to decrease methamphetamine-evoked DA re-
lease. Alone these analogs did not evoke DA release. UKMH-
106, but not UKMH-105, inhibited methamphetamine-
evoked DA release. Inhibition of DA uptake by the analogs at
the VMAT2 substrate site does not explain the C5 Z-selective
inhibition of the effect of methamphetamine on VMAT2.
UKMH-105 and UKMH-106 equipotently inhibited DA up-
take by VMAT2, but exhibited C5 Z-selective inhibition of
methamphetamine-evoked DA release, suggesting that these
two geometrical isomers interact with different sites on
VMAT2 to inhibit DA uptake and methamphetamine-evoked
DA release. Only, the Z double-bond geometry at the C5
position of the piperidine ring (UKMH-106) was tolerated by
the DA release site, whereas the DA uptake site also toler-
ated the C5 E geometry (UKMH-105). Thus, the VMAT2 site
mediating methamphetamine-evoked DA release is re-
stricted in its ability to accommodate both geometrical iso-
mers compared with the VMAT2 uptake site.
Although the mechanism by which methamphetamine re-
leases DA from synaptic vesicles is not understood fully,
potential mechanisms include weak base effects of metham-
phetamine, which disrupt vesicular proton gradients and
methamphetamine effects at the VMAT2 substrate site (Sul-
zer et al., 2005). Although having different double-bond ge-
ometries, UKMH-105 and UKMH-106 are expected to have
comparable pKavalues, inconsistent with the weak base hy-
pothesis as an explanation for differential effects in decreas-
ing methamphetamine-evoked DA release. However, current
observations are consistent with previous reports showing
differential effects of the amphetamine optical isomers (Ar-
nold et al., 1977; Fischer and Cho, 1979), despite having
identical pKavalues, which again does not support the weak
base hypothesis (Sulzer et al., 2005). Thus, UKMH-106 may
inhibit methamphetamine-evoked DA release through an in-
teraction with VMAT2 and not via a weak-base mechanism.
One caveat of the current study is that inhibitory effects of
the analogs on DA uptake and methamphetamine-evoked DA
release were evaluated using different preparations (i.e., iso-
lated vesicles and more intact slices, respectively). One alter-
native explanation is that the analogs may inhibit metham-
phetamine-evoked DA release by interacting with DAT in the
slice. Cytosolic DA is transported to the extracellular com-
partment through a methamphetamine-induced reversal of
DAT (Fischer and Cho, 1979). However, UKMH-106 inhib-
ited methamphetamine-evoked DA release 18-fold more po-
tently than inhibition of DAT function, making it unlikely
that inhibition of DAT is responsible for the decrease in
methamphetamine-evoked DA release. If inhibition of DAT
was responsible, then both UKMH-105 and UKMH-106
would be expected to decrease methamphetamine-evoked DA
release, because they are equipotent inhibiting DAT.
A concern regarding the approach of developing VMAT2
inhibitors as treatments for methamphetamine abuse is the
potential for neurotoxicity, because increased cytosolic DA
levels can lead to oxidative stress. Methamphetamine inhib-
its DA uptake at VMAT2, promotes DA release from vesicles,
inhibits monoamine oxidase, and produces DA deficits
caused by increased formation of reactive oxygen species
(Fleckenstein et al., 2007). To the contrary, lobeline protects
against methamphetamine-induced neurotoxicity (Eyerman
and Yamamoto, 2005). Furthermore, methamphetamine-ad-
dicted individuals given lobeline in phase I clinical trials
exhibited no adverse effects (http://www.clinicaltrials.gov/
tetrabenazine (a classic VMAT2 inhibitor) is approved by the
Food and Drug Administration for the treatment of Hunting-
ton’s chorea (Frank, 2010). Thus, precedent for the clinical use
of VMAT2 inhibitors exists. Nevertheless, evaluation of the
potential neurotoxicity of these analogs using animal models
will be an integral component of the drug development process
for these candidate treatments for methamphetamine abuse.
In summary, the current results extend our previous re-
search by showing that MTD decreases methamphetamine
self-administration without altering food-maintained re-
sponding, demonstrating that inhibition of VMAT2 function
translates to a promising behavioral result. However, MTD
has relatively low water solubility and diminishing drug
likeness and has high affinity (100 nM) for DAT, which may
result in abuse liability. Current results show that incorpora-
tion of the phenylethylene moiety of MTD into the piperidine
ring system, and the addition of aromatic dichloro substituents,
results in a novel candidate compound, UKMH-106, which has
improved water solubility and reduced affinity for DAT, SERT,
and nAChRs, thereby increasing selectivity for VMAT2. More-
over UKMH-106 decreased the effect of methamphetamine to
evoke DA release. Thus, the current research using a classic
that shows promise as a pharmacotherapy to treat metham-
phetamine abuse, a devastating problem for which there are no
Participated in research design: Horton, Hojahmat, Bardo, Crooks,
Conducted experiments: Horton, Siripurapu, Norrholm, Beck-
mann, Harrod, and Deaciuc.
Horton et al.
Contributed new reagents or analytic tools: Culver, Hojahmat, and
Performed data analysis: Horton, Siripurapu, Norrholm, Culver,
Hojahmat, Beckmann, Harrod, Deaciuc, Crooks, and Dwoskin.
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Address correspondence to: Dr. Linda Dwoskin, College of Pharmacy,
University of Kentucky, Lexington, KY 40536-0082. E-mail: ldwoskin@
MTD Analogs, Methamphetamine, and VMAT2