Effects of two
novel D3-selective compounds, NGB 2904
[N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide]
and CJB 090
[N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-4-(pyridin-2-yl)benzamide],
on the reinforcing and discriminative stimulus
effects of cocaine in rhesus
monkeys
Center for the
Neurobiological Investigation of Drug Abuse,
Department of Physiology and Pharmacology, Wake
Forest University School of Medicine,
NWinston-Salem, USA.
Abstract
The present study examined the effects of
two novel dopamine D3 receptor compounds, NGB
2904
[N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide],
an antagonist, and CJB 090
[N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-4-(pyridin-2-yl)benzamide],
a partial agonist, in two models of cocaine
abuse in rhesus monkeys. To establish a dose
range and time course of effects, both compounds
were shown to block quinpirole-induced yawning
when administered i.m. 15, 30, or 120 min before
quinpirole. Next, rhesus monkeys were trained to
discriminate i.m. injections of saline (0.5 ml)
and cocaine (0.3 mg/kg). Neither D3 compound
(0.03-3.0 mg/kg; n=3) substituted for cocaine in
any monkey. When given in combination with
cocaine, CJB 090 but not NGB 2904 attenuated the
discriminative stimulus effects of cocaine,
shifting the cocaine dose-response curve to the
right. In a separate group of monkeys,
responding was maintained under a second-order
schedule of either food (1.0-g pellets; n=3) or
cocaine (0.1 mg/kg/injection; n=4) presentation.
When responding was stable, a dose of NGB 2904
(1.0-5.6 mg/kg i.v.) or CJB 090 (0.3-3.0 mg/kg
i.v.) was administered for 5 consecutive days,
immediately before the session. CJB 090, but not
NGB 2904, decreased cocaine- and food-maintained
responding. These data indicate that compounds
with relatively high affinity and selectivity
for the D3 receptor can attenuate the
discriminative and reinforcing stimulus effects
of cocaine while not producing cocaine-like
effects. The present findings support the
continued examination of D3 compounds as
pharmacological tools for better understanding
the role of this receptor subtype in cocaine
addiction and as potential lead compounds for
novel
Cocaine abuse is a major problem in the
United States and worldwide [National
Institute on Drug Abuse (NIDA), 2004; World
Health Organization, 2004]. In 2002 alone,
there were an estimated 1.1 million new users of
cocaine, and the 2003 National Survey on Drug
Use and Health reported that 34.9 million
Americans 12 years of age and older confirmed
using cocaine at least once in their lifetime
(Substance Abuse and Mental Health Services
Administration, 2004). Despite over 30 years of
research, there is no safe and effective
pharmacotherapy to offer those afflicted by
cocaine addiction (Mello and Negus, 1996;
Carroll et al., 1999; Platt et al., 2002),
although several clinical trials are currently
underway (Gorelick et al., 2004; O'Brien, 2005;
Vocci et al., 2005). The dopamine (DA) system is
thought to play a primary role in the behavioral
and reinforcing effects of cocaine (Ritz et al.,
1987). Within the DA system, two superfamilies
of receptors have been identified, D1- and
D2-like receptors, and preclinical data suggest
that both are involved in the behavioral actions
of cocaine (e.g., Caine et al., 2000). The D3
receptor subtype belongs to the D2-like
superfamily of receptors and coexists with D2
receptors in mesolimbic areas of the brain
(Sokoloff et al., 1990). Because D3 receptors
are primarily localized in limbic brain regions,
compounds selectively blocking D3 receptors may
be free of extrapyramidal effects (Sokoloff et
al., 1990; Levant, 1997).
Furthermore, autoradiographic studies have
demonstrated higher densities of D3 receptors in
cocaine overdose victims compared with
noncocaine-abusing controls (Staley and Mash,
1996). Taken together, these studies support a
hypothesis that D3 receptors may be an important
therapeutic target in cocaine abuse.
Pharmacological tools provide evidence for a
possible role of D3 receptors in the
discriminative and reinforcing effects of
cocaine (e.g., Caine and Koob, 1993).
Pretreatments with the D2/D3 agonists quinpirole
or 7-hydroxy-N,N-di-n-propyl-2- aminotetralin
produced dose-dependent decreases in cocaine
self-administration in rats (Caine and Koob,
1993). More recently, the D3-selective partial
agonist BP 897 has been shown to decrease
cocaine seeking by rats self-administering
cocaine under a second-order schedule (Pilla et
al., 1999). Unlike the full agonists, BP 897
does not maintain selfadministration in rats or
monkeys (Pilla et al., 1999; Beardsley et al.,
2001) or mimic the discriminative stimulus
effects of cocaine or methamphetamine in monkeys
(Beardsley et al., 2001). One goal of the
present study was to examine, in rhesus monkeys,
another D3 partial agonist, CJB 090, in cocaine
discrimination and cocaine self-administration
studies.
D2-like receptor antagonists block
discriminative and reinforcing effects of
cocaine in rodents and monkeys (e.g., Kleven et
al., 1990; Caine et al., 2000). Advances in
medicinal chemistry have resulted in development
of D3-selective antagonists (Newman et al.,
2005). In a rodent model of relapse, the D3
antagonists SB-277011-A and NGB 2904 inhibited
cocaine-induced drug seeking (Gilbert et al.,
2005; Gal and Gyertyan, 2006). Also in rodents,
SB-277011-A decreased cocaine
self-administration under several conditions
(Vorel et al., 2002; Di Ciano et al., 2003; Xi
et al., 2005, 2006). A second goal of the
present study was to assess the ability of
another D3-selective antagonist, NGB 2904, to
alter the discriminative and reinforcing effects
of cocaine in rhesus monkeys.
The D3 antagonist NGB 2904 binds with
approximately 56-fold selectivity at the human
D3 receptor (hD3) compared with the human D2L
receptor (hD2L), whereas the D3 partial agonist
CJB 090 has approximately 50-fold selectivity at
the hD3 compared with the hD2L receptor (Grundt
et al., 2005). Both NGB 2904 and CJB 090 are
twice as selective for hD3 receptors as is
quinpirole, which has 23-fold selectivity for D3
over D2 receptors in HEK 293 cells (R. Luedtke,
unpublished data). Because neither drug had been
evaluated in nonhuman primates, the first
experiment examined their ability to block
quinpirole-induced yawning, suggested by
behavioral experiments in rodents to be D3
receptor mediated (Collins et al., 2005). The
second experiment examined the ability of each
compound to substitute for cocaine and to shift
the cocaine dose-response curve in monkeys
trained to discriminate cocaine. As a positive
control, we also evaluated the nonselective
D2/D3 antagonist haloperidol in these same
monkeys. The third experiment assessed the
ability of NGB 2904 and CJB 090 to alter
cocaine- and food-reinforced responding using a
second-order schedule that has been shown to be
sensitive to D3 partial agonist effects (Pilla
et al., 1999).
Discussion
The effects of two novel DA D3-selective
compounds were evaluated in several nonhuman
primate models. Both the D3-selective partial
agonist CJB 090 and the D3-selective antagonist
NGB 2904 dose-dependently reversed
quinpiroleinduced yawning. Neither compound
substituted for cocaine in drug discrimination
studies. CJB 090 blocked the discriminative
stimulus effects of cocaine and decreased
cocaine- and food-maintained responding. In
contrast, NGB 2904 produced equivocal effects on
cocaine discrimination and did not affect rates
of cocaine- or food-maintained responding at
doses that reversed quinpirole-induced yawning.
Neither drug produced D2-like side effects, such
as catalepsy or stereotypy, at doses that
affected cocaine discrimination and
self-administration. The current findings
further support the continued development of
compounds with high affinity at D3 receptors as
potential treatment agents for cocaine abuse.
There is growing interest in the use of D3
compounds in preclinical models of cocaine abuse
(see Newman et al., 2005). Partial agonists may
provide a unique versatility as a potential
pharmacotherapeutic treatment because the
efficacy of the compounds should vary with
levels of extracellular DA competing for the
receptor (Pulvirenti and Koob, 1994; Childress
and O'Brien, 2000; Platt et al., 2002; Negus,
2006). In contrast, an antagonist would be
expected to decrease cocaine seeking and
decrease the positive subjective effects of
cocaine, irrespective of levels of extracellular
DA (Platt et al., 2002). Because CJB 090 and NGB
2904 had not previously been tested in monkeys,
we first assessed the effects of both drugs on
an unconditioned behavior believed to be
mediated by D3 receptors based on studies in
rodents (see Collins et al., 2005).
Extending earlier results in rats, the D2/D3
agonist quinpirole was shown to significantly
induce yawning in monkeys in a dose-dependent
fashion. Examining several D2/D3 compounds on
quinpirole-induced yawning, Collins et al.
(2005) concluded that the ascending limb of the
quinpirole dose-response curve was mediated via
D3 receptors, whereas the descending limb was
mediated via D2 receptors. At the peak of the
quinpirole dose-response curve, D3- but not
D2-selective antagonists significantly reduced
quinpirole-induced yawning (Collins et al.,
2005). In the present study, when tested in
combination with the dose of quinpirole that
induced maximal yawning, both CJB 090 and NGB
2904 dose-dependently attenuated yawning; both
compounds had similar potency, supporting the
hypothesis that these drugs are acting as
dopamine D3 receptor antagonists in vivo.
Further investigation of the pharmacological
basis of quinpirole-induced yawning in monkeys,
as observed in rodents (Collins et al., 2005),
will require additional testing and is beyond
the scope of the present report. Nevertheless,
in addition to confirming in vivo activity in
monkeys, these results provide important time
course data related to the drug discrimination
and self-administration studies. In vitro
studies demonstrated that CJB 090 was 50-fold
selective for D3 receptors over D2 receptors in
human D3 and D2 receptor transfected cell lines
(Grundt et al., 2005). There seems to be some
species differences because CJB 090 is nearly
100-fold selective for D3 receptors over D2
receptors in rat-transfected cell lines (Newman
et al., 2003). Functional in vitro assays of
agonist-induced mitogenesis established that CJB
090 induced only up to a 29.7% increase in
mitogenesis, suggesting that CJB 090 functioned
as a D3-selective partial agonist (Newman et
al., 2003).
However, CJB 090 did not show a behavioral
profile that was indicative of a D3 agonist
because it neither elicited significant yawning
when administered alone nor substituted for
cocaine in drug discrimination. This is similar
to what has been reported with BP 897, a
purported D3 partial agonist, which does not
have cocaine-like discriminative stimulus
effects or maintain selfadministration in rats
and monkeys (Beardsley et al., 2001). Taken
together, these findings suggest that
identification of partial agonists using in
vitro functional assays may not predict
agonist-like activity in vivo. However, when
evaluated in combination with cocaine, CJB 090
was able to attenuate the
discriminative-stimulus effects of cocaine,
resulting in rightward shifts in the cocaine
dose-response curve. Attenuation of the cocaine
cue by CJB 090 was surmountable by increasing
doses of cocaine. When tested in monkeys
selfadministering cocaine under a second-order
schedule, CJB 090 also decreased
cocaine-maintained responding. Doses of CJB 090
that decreased cocaine self-administration also
decreased responding maintained by food,
suggesting nonselective reductions in cocaine
reinforcement (but see Nader et al., 2002).
Likewise, BP 897 has been shown to reduce
cue-induced reinstatement in rats, to attenuate
the discriminative stimulus effects of cocaine
in mice, and to decrease cocaine seeking in rats
responding under a second-order schedule (Pilla
et al., 1999; Beardsley et al., 2001; Cervo et
al., 2003; Gilbert et al., 2005). Taken
together, these data suggest that in the
presence of high dopaminergic tone, i.e., after
cocaine administration, DA D3 receptors play a
modulatory role in the production of
cocaine-induced interoceptive cues and in the
positive reinforcing effects of cocaine. NGB
2904 has been classified as an antagonist at DA
D3 receptors with high binding affinity (1.4 nM)
at the hD3 receptor and 150-fold selectivity for
primate D3 receptors over primate D2L receptors
(Yuan et al., 1998). As was noted with CJB 090,
species variation has been observed with NGB
2904. For example, NGB 2904 showed 830-fold
selectivity for D3 over D2 receptors in cloned
rat DA receptors and only 56-fold selectivity in
cloned human DA receptors (Newman et al., 2003;
Grundt et al., 2005). Irrespective of these
binding affinities, NGB 2904 selectively and
potently inhibited D3 receptors as demonstrated
by antagonism of quinpirole-stimulated
mitogenesis (Yuan et al., 1998), and it
decreased quinpirole-induced yawning in monkeys
(present study). In addition, NGB 2904
injections did not maintain self-administration
in monkeys (J. L. Martelle, J. T. Ross, and M.
Nader, unpublished data) and did not substitute
for cocaine in drug discrimination, further
suggesting a lack of agonist action.
In the present study, NGB 2904 had equivocal
effects on cocaine discrimination and was
inactive in decreasing cocaine- maintained
responding under a second-order schedule. Doses
that were effective in blocking
quinpirole-induced yawning did not affect
cocaine self-administration in rhesus monkeys.
In an earlier study using the moderately
selective D3 antagonist PNU 99194-A, which has
an approximate 100- fold lower affinity at D3
receptors compared with NGB 2904, decreases in
cocaine- and food-maintained responding were
observed (Claytor et al., 2006). The fact that
we did not observe significant effects on
cocaine self-administration following NGB 2904
administration under conditions in which PNU
99194-A did affect responding suggests that NGB
2904, despite its higher affinity and
selectivity for D3 receptors, may not be an
effective pharmacotherapy for cocaine abuse.
However, it should be pointed out that D3
receptor antagonists have shown promise in
rodent models of cocaine abuse (Ashby et al.,
2003; Di Ciano et al., 2003; Gilbert et al.,
2005; Xi et al., 2005, 2006). Previous
experiments have demonstrated that higher doses
of NGB 2904 produce diminished D3
antagonist-like effects compared with
intermediate doses (Gilbert et al., 2005). This
finding could be related to the high
lipophilicity of NGB 2904 leading to low
bioavailability, poor pharmacokinetics, and
significantly lower absolute levels in the brain
after i.v. administration compared with another
D3-selective antagonist, SB-277011-A (Reavill et
al., 2000), under the same conditions (Newman et
al., 2005). Another possibility for the
equivocal effects of NGB 2904 on cocaine
discrimination, but not CJB 090, may be related
to the affinity of each compound at D2 receptors
(Ki 112 versus 24 nM for NGB 2904 and CJB 090,
respectively; Grundt et al., 2005). However,
there was no evidence of catalepsy induced by
any dose of CJB 090 or NGB 2904, which suggests
that D2 receptors are not likely involved in the
behaviors observed in this study. Nevertheless,
the precise contribution of D2 and D3 receptors
in the behavioral effects of NGB 2904, CJB 090
and other "D3-selective" compounds will require
additional studies with highly selective D2 and
D3 agonists, which are not currently
available.
The present study aimed to examine: 1) the
effects of a potent and selective D3 antagonist
(NGB 2904) and partial agonist (CJB 090) on the
reinforcing and discriminative stimulus effects
of cocaine in rhesus monkeys and 2) whether or
not a D3 partial agonist, as determined by
quinpirole-stimulated mitogenesis in Chinese
hamster ovary cells, would show any agonist
activity in vivo. In summary, we found that the
D3 partial agonist CJB 090, but not the D3
antagonist NGB 2904, was able to block the
discriminative stimulus effects of cocaine and
to decrease cocaine self-administration. There
was no evidence that CJB 090 produced DA agonist
effects in vivo. It remains possible that the
behavioral effects observed with CJB 090 were
due to D2 receptor antagonism, although blockade
of the behavioral effects of cocaine occurred at
doses that did not induce catalepsy. These
findings support the continued examination of
compounds acting at D3 receptors as
pharmacological tools for further understanding
the mechanistic underpinnings of cocaine
addiction and as potential leads for therapeutic
agents.
