Alanine analogues of [D-Trp]CJ-15,208: Novel opioid activity profiles and prevention of drug- and stress-induced reinstatement of cocaine-seeking behaviour

Article abstract of DOI:10.1111/bph.12664

Background and Purpose The novel macrocyclic peptide cyclo[Phe-D-Pro-Phe-D- Trp] ([D-Trp]CJ-15,208) exhibits κ opioid (KOP) receptor antagonist activity in both in vitro and in vivo assays. The four alanine analogues of this peptide were synthesized and characterized both in vitro and in vivo to assess the contribution of different amino acid residues to the activity of [D-Trp]CJ-15,208. Experimental Approach The peptides were synthesized by a combination of solid phase peptide synthesis and cyclization in solution. The analogues were evaluated in vitro in receptor binding and functional assays, and in vivo with mice using a tail-withdrawal assay for antinociceptive and opioid antagonist activity. Mice demonstrating extinction of cocaine conditioned-place preference (CPP) were pretreated with selected analogues to evaluate prevention of stress or cocaine-induced reinstatement of CPP. Key Results The alanine analogues displayed pharmacological profiles in vivo distinctly different from [D-Trp]CJ-15,208. While the analogues exhibited varying opioid receptor affinities and κ and μ opioid receptor antagonist activity in vitro, they produced potent opioid receptor-mediated antinociception (ED₅₀ = 0.28-4.19-nmol, i.c.v.) in vivo. Three of the analogues also displayed KOP receptor antagonist activity in vivo. Pretreatment with an analogue exhibiting both KOP receptor agonist and antagonist activity in vivo prevented both cocaine- and stress-induced reinstatement of cocaine-seeking behaviour in the CPP assay in a time-dependent manner. Conclusions and Implications These unusual macrocyclic peptides exhibit in vivo opioid activity profiles different from the parent compound and represent novel compounds for potential development as therapeutics for drug abuse and possibly as analgesics.

Full text of DOI:10.1111/bph.12664

DOI:10.1111/bph.12664
www.brjpharmacol.org
British Journal of
Pharmacology
BJP
Correspondence
RESEARCH PAPER
Jane V Aldrich, Department of
Medicinal Chemistry, University
of Kansas, 1251 Wescoe Hall Dr,
Lawrence, KS 66045, USA. E-mail:
jaldrich@ku.edu
Alanine analogues of
[D-Trp]CJ-15,208: novel
opioid activity profiles and
prevention of drug- and
stress-induced reinstatement
of cocaine-seeking
----------------------------------------------------------------
*Present address: The Scripps
Research Institute, Jupiter, FL,
USA.
----------------------------------------------------------------
Keywords
CJ-15,208; macrocyclic peptide; κ
opioid receptor antagonist;
analgesic; cocaine reinstatement
----------------------------------------------------------------
Received
24 October 2013
Revised
30 January 2014
Accepted
24 February 2014
behaviour
J V Aldrich1, S N Senadheera1, N C Ross1*, K A Reilley2, M L Ganno2,
S E Eans2, T F Murray3 and J P McLaughlin2
1Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS, USA,
2Department of Biology, Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL, USA, and
3Department of Pharmacology, Creighton University School of Medicine, Omaha, NE, USA
BACKGROUND AND PURPOSE
The novel macrocyclic peptide cyclo[Phe-D-Pro-Phe-D-Trp] ([D-Trp]CJ-15,208) exhibits κ opioid (KOP) receptor antagonist
activity in both in vitro and in vivo assays. The four alanine analogues of this peptide were synthesized and characterized both
in vitro and in vivo to assess the contribution of different amino acid residues to the activity of [D-Trp]CJ-15,208.
EXPERIMENTAL APPROACH
The peptides were synthesized by a combination of solid phase peptide synthesis and cyclization in solution. The analogues
were evaluated in vitro in receptor binding and functional assays, and in vivo with mice using a tail-withdrawal assay for
antinociceptive and opioid antagonist activity. Mice demonstrating extinction of cocaine conditioned-place preference (CPP)
were pretreated with selected analogues to evaluate prevention of stress or cocaine-induced reinstatement of CPP.
KEY RESULTS
The alanine analogues displayed pharmacological profiles in vivo distinctly different from [D-Trp]CJ-15,208. While the
analogues exhibited varying opioid receptor affinities and κ and μ opioid receptor antagonist activity in vitro, they produced
potent opioid receptor-mediated antinociception (ED50 = 0.28–4.19 nmol, i.c.v.) in vivo. Three of the analogues also displayed
KOP receptor antagonist activity in vivo. Pretreatment with an analogue exhibiting both KOP receptor agonist and antagonist
activity in vivo prevented both cocaine- and stress-induced reinstatement of cocaine-seeking behaviour in the CPP assay in a
time-dependent manner.
CONCLUSIONS AND IMPLICATIONS
These unusual macrocyclic peptides exhibit in vivo opioid activity profiles different from the parent compound and represent
novel compounds for potential development as therapeutics for drug abuse and possibly as analgesics.
© 2014 The British Pharmacological Society
3212 British Journal of Pharmacology (2014) 171 3212–3222

Opioid profiles of [D-Trp]CJ-15,208 Ala analogues
BJP
Abbreviations
CHO, Chinese hamster ovary; CJ-15,208, cyclo[Phe-D-Pro-Phe-Trp]; CPP, conditioned-place preference; DAMGO,
[D-Ala2,NMePhe4,glyol5]enkephalin; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DOP receptor, δ opioid
receptor; DPDPE, cyclo[D-Pen2,D-Pen5]enkephalin (Pen = penicillamine); [D-Trp]CJ-15,208, cyclo[Phe-D-Pro-Phe-D-Trp];
Dyn, dynorphin; β-FNA, β-funaltrexamine; GNTI, 5′-guanidinylnaltrindole; JDTic, (3R)-7-hydroxy-N-((1S)-1-[[(3R,4R)-4-
(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide;
KOP receptor, κ opioid receptor; KOP KO, KOP receptor gene-disrupted mice; MOP receptor, μ opioid receptor; MOP
KO, MOP receptor gene-disrupted mice; nor-BNI, nor-binaltorphimine; SNC-80, (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-
dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; WT, wild type; U50,488, ( )-trans-3,4-dichloro-
N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide
ing the structure–activity relationships of both Trp isomers of
CJ-15,208. To determine which amino acid side chains were
Introduction
important for the observed opioid activity, each amino acid
in CJ-15,208 was initially replaced by alanine (Aldrich et al.,
2011). Unexpected differences in their opioid activity in vivo
Evidence suggests that κ opioid (KOP) receptor antagonists
may have therapeutic potential in the treatment of drug
abuse (Aldrich and McLaughlin, 2009). For example, pre-
were found for the alanine analogues and CJ-15,208 com-
treatment with the KOP receptor-selective small-molecule
pared with the results obtained in vitro. Therefore, we also
antagonists nor-binaltorphimine (nor-BNI) and ((3R)-7-
examined the alanine-substituted analogues of cyclo[Phe-D-
hydroxy-N-((S)-1-[[(3R,4R)-4-(3-hydroxyphenyl)-3,4-
Pro-Phe-D-Trp] ([D-Trp]CJ-15,208; Figure 1B) both in vitro and
dimethyl-1-piperidinyl]methyl]-2-methylpropyl)-1,2,3,
4-tetrahydro-3-isoquinolinecarboxamide) (JDTic) has been
shown to prevent the stress-induced reinstatement of
extinguished cocaine-seeking behaviour (Beardsley et al.,
in vivo to determine the contribution of each residue to this
parent peptide’s opioid receptor interactions and its opioid
activity profile in vivo.
2005; Redila and Chavkin, 2008), and heroin-dependent
Statement on drug and receptor nomenclature
patients treated for 12 weeks with a ‘functional KOP receptor
All drug and molecular target terms conform to parameters
antagonist’ (buprenorphine plus naltrexone) showed signifi-
specified in the British Journal of Pharmacology’s Guide to
Receptors and Channels (Alexander et al., 2013).
cantly improved drug abstinence relative to patients treated
only with naltrexone (Rothman et al., 2000; Gerra et al.,
2006). However, the prototypical KOP receptor-selective
non-peptide antagonists nor-BNI, 5′-guanidinylnaltrindole
(GNTI) and JDTic exhibit exceptionally long activity, antago-
Methods
nizing KOP receptors for weeks after a single dose (Metcalf
Materials
and Coop, 2005; Aldrich and McLaughlin, 2009). This profile
The sources of reagents, amino acids, 2-chlorotrityl chloride
could potentially complicate their use as pharmacological
resin and solvents are the same as reported previously (Ross
tools and as possible therapeutic agents, spurring the search
et al., 2010; Aldrich et al., 2011). Amino acids are the L-isomer
for shorter acting KOP receptor-selective antagonists (Brugel
unless otherwise specified and abbreviations for amino acids
et al., 2010; Runyon et al., 2010; Grimwood et al., 2011; Peters
follow the IUPAC-IUB Joint Commission of Biochemical
et al., 2011; Frankowski et al., 2012).
In addition to dynorphin A derivatives (Bennett et al.,
2002; Patkar et al., 2005), we are examining novel peptides
unrelated to the endogenous opioid peptides as KOP receptor
antagonists. Of particular interest was the novel tetrapeptide
CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) that was reported to
exhibit KOP receptor antagonist activity in vitro (Saito et al.,
2002). The relatively low molecular weight (577 Da) and mac-
rocyclic structure of CJ-15,208 suggested it would be a prom-
ising lead candidate for potential development. Macrocyclic
tetrapeptides are stable to proteolytic degradation (Delaforge
et al., 1997), so it was expected that these peptides would
exhibit activity in vivo after systemic administration.
Because the stereochemistry of the Trp residue in
CJ-15,208 was not determined when this natural product was
isolated, we synthesized both tryptophan isomers of this
macrocyclic peptide (Kulkarni et al., 2009; Ross et al., 2010),
and found that the peptide containing L-Trp was the natural
product based on its optical rotation. The D-Trp containing
peptide (Figure 1A) also preferentially bound to KOP recep-
Figure 1
tors and exhibited antagonist activity at these receptors, both
in vitro (Dolle et al., 2009; Ross et al., 2010; 2012) and in vivo
(Ross et al., 2012; Eans et al., 2013). Therefore, we are explor-
Structures of (A) [D-Trp]CJ-15,208 (1) and (B) alanine analogues 2–5.
The residues are numbered 1–4, arbitrarily starting with the Phe
C-terminal to the D-Trp residue.
British Journal of Pharmacology (2014) 171 3212–3222 3213

J V Aldrich et al.
BJP
Nomenclature [Eur J Biochem (1984) 138: 9–37]. All chemi-
cals other than analogues of [D-Trp]CJ-15,208 were obtained
from Sigma-Aldrich (St. Louis, MO, USA). [D-Trp]CJ-15,208
and synthesized analogues (Figure 1) were dissolved daily
prior to administration initially in dimethyl sulfoxide
(DMSO), and sufficient warm (40°C) sterile saline (0.9% )
then added so that the final vehicle for in vivo administration
consisted of one part DMSO and one part sterile saline.
[3H]diprenorphine, [3H]-[D-Ala2,NMePhe4,glyol5]enkephalin
([3H]DAMGO) [3H]cyclo[D-Pen2,D-Pen5]enkephalin
and
([3H]DPDPE) respectively. IC50 values were determined by non-
linear regression analysis using Prism software (GraphPad
Software Co., La Jolla, CA, USA). Ki values were calculated from
the IC50 values by the Cheng and Prusoff equation (Cheng and
Prusoff, 1973) using KD values of 0.45, 0.49 and 1.76 nM for
[3H]diprenorphine, [3H]DAMGO and [3H]DPDPE respectively.
These results are presented as the mean SEM from at least
three separate experiments each performed in triplicate.
Peptide synthesis and purification
The linear peptide precursors (based on the parent sequence
H-D-Trp-Phe-D-Pro-Phe-OH with substitution of D-Ala, Ala,
D-NMeAla and Ala in positions 1–4, respectively) were synthe-
sized on a 2-chlorotrityl choride resin by Fmoc solid phase
synthesis, and the peptides cleaved from the resin with 1%
trifluoroacetic acid in dichloromethane as described previ-
ously (Ross et al., 2010; Aldrich et al., 2011). The crude linear
peptides were used in the cyclization reactions without further
purification. The peptides were initially cyclized using the
previously reported procedure (Ross et al., 2010; Aldrich et al.,
2011). This procedure was subsequently modified as follows to
increase the yields of the cyclic peptides (Senadheera et al.,
2011): the crude linear peptide (1 equiv, 0.62 mM in 20 mL
N,N-dimethylformamide, DMF) was added dropwise at a rate
of 1.4 mL·h−1 (using a KD Scientific single infusion syringe
pump, Lab Source Inc., Romeoville, IL, USA) to a dilute
solution of HATU [2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate, 1.5 equiv,
0.938 mM] and N,N-diisopropylethylamine (8 equiv, 5 mM)
in DMF. After 15 h additional HATU (1.5 equiv) was then
added to the reaction in one portion, and additional linear
peptide (1 equiv, 0.62 mM in 20 mL DMF) was added dropwise
at a rate of 1.2 mL·h−1 as described above. The reaction was
then stirred for 12 h at room temperature, followed by an
additional 20–24 h at 37°C. The solvent was evaporated under
reduced pressure, and the crude cyclic tetrapeptides isolated as
previously described (Ross et al., 2010; Aldrich et al., 2011).
Initially, the peptides were purified by reversed-phase
HPLC as described previously (Ross et al., 2010; Aldrich et al.,
2011). Larger quantities of peptides for in vivo evaluation were
purified by silica gel chromatography using a step gradient of
60–90% EtOAc in hexane (with EtOAc increased in 10%
increments), followed by 0–3% MeOH in EtOAc (with MeOH
increased in 1% increments). For the more polar analogues 2
and 4, the gradient used was 0–3% MeOH in EtOAc. The
purified peptides were dissolved in aqueous acetonitrile
(water : MeCN, 4:1) and then lyophilized to give the peptides
as white solids. The yields of the Ala analogues after purifi-
cation were 45–55%.
GTPγS assays. The binding of [35S]GTPγS to membranes from
CHO cells stably expressing KOP or MOP receptors was
assayed as described previously (Siebenaller and Murray,
1999; Aldrich et al., 2011; Ross et al., 2012). Efficacy was
determined relative to the reference full agonists dynorphin
(Dyn) A-(1–13) amide for KOP receptors and DAMGO for
MOP receptors. The antagonist profiles of the peptides at KOP
and MOP receptors were determined by measuring the EC50
values of Dyn A-(1–13)amide and DAMGO, respectively, in
the absence and presence of four different concentrations
(0.1 nM−10 μM) of the peptide, performed in triplicate. The
pA2 values were determined by Schild analysis (Schild, 1947),
and the results are reported as KB values SEM from at least
three experiments except where noted.
In vivo pharmacological evaluation
Animals and drug administration. All animal care and experi-
mental procedures complied with the National Institute of
Health Guide for the Care and Use of Laboratory Animals and
were approved by the Institutional Animal Care and Use
Committee at the Torrey Pines Institute for Molecular Studies.
All results of animal testing are reported in accordance with
the ARRIVE guidelines (Kilkenny et al., 2010; McGrath et al.,
2010). A total of 852 animals were used in the experiments
described here.
Adult male wild-type C57BL/6J mice weighing 20–25 g
were obtained from Jackson Laboratory (Bar Harbor, ME,
USA). MOP receptor gene-disrupted (MOP KO) and KOP
receptor gene-disrupted (KOP KO) mice were obtained from
colonies established at the Torrey Pines Institute for Molecu-
lar Studies from homozygous breeding pairs of mice back-
crossed to C57BL/6J inbred mice for at least 12 generations
and obtained from the Jackson Laboratory. All mice were kept
on a 12 h light–dark cycle and food pellets and distilled water
were available ad libitum.
Antinociceptive testing. The 55°C warm-water tail-withdrawal
assay was performed in mice as previously described
(McLaughlin et al., 1999), with the latency of the mouse to
withdraw its tail from the water taken as the end point (a
cut-off time of 15 s was used in this assay). Peptides were
administered by i.c.v. injection as described previously
(Aldrich et al., 2011). Antinociception was calculated accord-
ing to the following formula: % antinociception = 100× (test
latency − control latency)/(15 − control latency). Tail with-
drawal data points are the means of 8–12 mice, unless other-
wise indicated, with SEM shown by error bars. Tail withdrawal
latencies in KO mice were determined in six to eight mice.
To determine the opioid receptor involvement in the
agonist activity of macrocyclic peptides 2–5, mice were
The purified peptides were analysed by electrospray ioni-
zation mass spectrometry, thin-layer chromatography and
analytical HPLC (see Supporting Information Appendix S1).
All peptides were >99% pure in both HPLC systems.
In vitro pharmacological analysis
Radioligand binding assays. Opioid receptor affinities were
determined in radioligand binding assays as previously
described (Arttamangkul et al., 1997; Aldrich et al., 2011; Ross
et al., 2012) with membranes from Chinese hamster ovary
(CHO) cells stably expressing rat KOP, rat μ opioid (MOP)
or mouse δ opioid (DOP) receptors using the radioligands
3214 British Journal of Pharmacology (2014) 171 3212–3222

Opioid profiles of [D-Trp]CJ-15,208 Ala analogues
BJP
pretreated with a single dose of β-funaltrexamine (β-FNA,
experiments were evaluated with ANOVA using Tukey’s or Dun-
nett’s multiple comparison post hoc tests, as appropriate.
Analyses were used to compare baseline and post-treatment
tail-withdrawal latencies and to determine statistical signifi-
cance for all tail-withdrawal data. Statistical significance of
differences between ED50 values was determined by evalua-
tion of the ED50 value shift via non-linear regression mod-
eling with Prism 6.02. One-way ANOVA was performed on all in
vivo receptor agonist and antagonist selectivity data. Data for
CPP experiments were analysed by multivariate ANOVA with
the main effect of CPP phase (e.g. postconditioning, week of
preference test, reinstatement) and the interaction of drug
pretreatment (macrocyclic peptide or vehicle) and reinstate-
ment condition (stress or cocaine exposure). Significant (P <
0.05) effects were further analysed using Tukey’s honestly
significant difference (HSD) post hoc test.
5 mg·kg−1, s.c.) or nor-BNI (10 mg·kg−1, i.p.) 23.5 h in advance
of administration of
a dose of a macrocyclic peptide.
Additional mice were pretreated with a single dose of naltrin-
dole (20 mg·kg−1, i.p.) 15 min prior to administration of the
macrocyclic tetrapeptide.
To determine antagonist activity, mice were pretreated
with the macrocyclic tetrapeptide 140 min prior to the
administration of the MOP receptor-preferring agonist mor-
phine (10 mg·kg−1, i.p.), the KOP receptor-selective agonist
U50,488 (10 mg·kg−1, i.p.) or the DOP receptor-selective
agonist SNC-80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-
1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide,
100 nmol, i.c.v.); at this time the antinociceptive activity of
the macrocyclic peptides had dissipated. Antinociception
produced by these established agonists was then measured
40 min after their administration. Additionally, to determine
the duration of KOP receptor antagonist activity, other mice
were pretreated 2.3–23.3 h prior to administration of
U50,488 as described above.
Results
Synthesis
Conditioned-place preference (CPP) evaluation. Male C57BL/6J
mice (weighing 20–25 g at the beginning of the experiment)
were subjected to an unbiased and counterbalanced cocaine
CPP paradigm, where the compartment in which the animal
receives vehicle or drug is randomly assigned regardless of
initial preference, using similar timing as detailed previously
(Eans et al., 2013). The time the animals spent in the two outer
and middle compartments was measured for 30 min in boxes
outfitted with infrared beams (San Diego Instruments, San
Diego, CA, USA); data are reported as the difference in time
spent in the cocaine-minus the vehicle-paired compartment.
Mice were subjected to 4 days of place conditioning to
0.9% saline and cocaine (10 mg·kg−1, s.c. in 0.9% saline) as
previously described (Aldrich et al., 2009; Ross et al., 2012;
Eans et al., 2013). The mice were then evaluated for their
place preference for 30 min twice weekly over a 3 week period
until extinction was established (see Figure 8).
Following demonstration of extinction, groups of mice
(10–24) were exposed to either forced swim stress or an addi-
tional cycle of cocaine place conditioning (see Figure 8A) as
described previously (Carey et al., 2007; Aldrich et al., 2009;
Ross et al., 2012). Mice were pretreated with vehicle, macro-
cyclic peptide 2 (10 nmol, i.c.v.) or peptide 5 (30 nmol, i.c.v.)
on days 28 and 29 2 h prior to a forced swimming session on
each day, performed as previously described (Aldrich et al.,
2009; Ross et al., 2012; Eans et al., 2013). Additional mice
were treated for 2 days with vehicle or macrocyclic peptide
5 min, 30 min, 1 h or 2 h prior to an additional session of
cocaine conditioning on day 29 (Figure 8A). Mice were tested
for place preference on the day following completion of the
stress exposure or cocaine place conditioning (day 30, see
Figure 8A).
The alanine analogues of [D-Trp]CJ-15,208 (Figure 1) were
synthesized by a combination of solid phase synthesis of
the linear precursors followed by cyclization in solution
(Kulkarni et al., 2009; Ross et al., 2010). Modifications were
made to the cyclization reaction, namely increasing the reac-
tion temperature and decreasing the rate of addition of the
linear peptide to the reaction, to improve the yields of the
macrocyclic peptides (Senadheera et al., 2011; Aldrich et al.,
2013). Initially, the peptides were purified by reversed-phase
HPLC; subsequent purifications were performed by flash
chromatography on silica gel, which permitted the facile
purification of larger quantities of the macrocyclic peptides
for in vivo pharmacological evaluation.
In vitro pharmacological evaluation
The affinities of the alanine analogues of [D-Trp]CJ-15,208 for
KOP receptors varied substantially (Table 1). Analogue 2, in
which Phe1 was replaced by Ala (see Figure 1B for residue
numbering), exhibited the highest KOP receptor affinity, sev-
enfold higher than the parent macrocyclic peptide 1. In con-
trast, analogue 5, in which D-Trp4 was replaced by D-Ala,
exhibited very low KOP receptor affinity (Ki = 1.7 μM), sug-
gesting that the D-Trp4 residue was important for binding to
these receptors. Analogues 3 and 4 exhibited intermediate
affinities for KOP receptors, 3.5- and 7.7-fold lower than that
of analogue 1. Similar results were reported by Dolle et al.
(2009) for analogues 2 and 5, but the KOP receptor affinity of
analogue 4 found here (167 nM) is substantially higher than
that reported by Dolle et al. (2009) (Ki = 1300 nM). (Note that
analogue 3 has not been previously reported.)
MOP receptor affinity was generally less sensitive to
alanine substitution in the macrocyclic tetrapeptide than was
affinity for the KOP receptor (Table 1), although there was
some variation among the analogues. Substitution of Phe1 by
Ala to give 2 substantially increased MOP receptor affinity,
while substitution of D-Trp4 by D-Ala to give 5 decreased
affinity threefold. Replacement of D-Pro2 by D-NMeAla or of
Phe3 by Ala had little effect on MOP receptor affinity. Analo-
gous to the results for the KOP receptor, the MOP receptor
Data analysis. All data are presented as mean SEM. Graded
dose–response curves were constructed, and potencies are
reported as the effective dose producing 50% antinociception
(ED50). All dose–response lines were analysed by non-linear
regression, and ED50 values and 95% confidence intervals
determined using individual data points with the Prism 6.02
software package (GraphPad). Data for antinociception
British Journal of Pharmacology (2014) 171 3212–3222 3215

J V Aldrich et al.
BJP
Table 1
Opioid receptor affinities of the alanine analogues of [D-Trp]CJ-15,208
Ki (nM SEM)
Selectivity
Peptide
KOP receptor
MOP receptor
DOP receptor
KOP/MOP/DOP
2, Ala1
3, D-NMeAla2
4, Ala3
3.07 0.30
76.9 18.2
167 15
27.3 2.7
257 14
299 114
775 71
259 29
8330 1220
5690 610
>10 000
1/6.7/2620
1/3.3/74
1/1.8/>59
2.2/1/>13
1/12/192
5, D-Ala4
1720 420
21.8 4.8
>10 000
1
4190 858
CHO membranes expressing cloned KOP, MOP or DOP receptors were incubated with different concentrations of each macrocyclic
tetrapeptide in the presence of [3H]diprenorphine, [3H]DAMGO or [3H]DPDPE, for KIL KOP, MOP and DOP receptors, respectively. Data are
the mean Ki values SEM from at least three experiments.
MOP receptor antagonists also generally correlated with their
Table 2
respective MOP receptor affinities, except for the D-Ala4 ana-
Opioid antagonist activity of the alanine analogues in vitro in the
[
35S]GTPγS assaya
logue 5 that exhibited weaker antagonist activity than might
be expected based on its affinity.
Antagonism: KB (nM SEM)
In vivo pharmacological evaluation
Antinociceptive activity. The analogues were initially evalu-
ated for their antinociceptive activity in the 55°C warm-water
tail-withdrawal assay in C57BL/6J mice following i.c.v.
administration. The parent peptide 1 exhibited minimal anti-
nociceptive activity at doses up to 10 nmol (Ross et al., 2012),
with moderate antinociceptive activity (40%) detected only
at 30 nmol, i.c.v. (Figure 2A). In contrast, all of the alanine
analogues exhibited potent antinociceptive activity, which
was unexpected given the lack of efficacy observed for the
analogues in vitro in the GTPγS assay. Significant antinocicep-
tive activity was detected for 80–100 min after administration
of each analogue at their highest respective dose (Figure 2B).
The D-Ala analogue 5 produced potent antinociception [with
an ED50 (and 95% confidence interval) of 0.28 (0.15–
0.55) nmol, i.c.v.], which was particularly surprising given its
low affinity for opioid receptors. The Ala1 (2) and Ala3 (4)
analogues exhibited similar antinociceptive potencies in this
in vivo assay, with ED50 values of 1.99 (0.36–13.2) and 1.04
(0.45–2.47) nmol, i.c.v. respectively. The D-NMeAla analogue
(3) was the least potent of the alanine analogues [ED50 = 4.19
(1.40–11.9) nmol, i.c.v.]. Although the potencies differed, all
four analogues produced antinociception comparable with
Peptide
KOP receptor
MOP receptor
2, Ala1
3, D-NMeAla2
4, Ala3
0.81 0.58
103 38
8.08 2.44
321 186
1470 160b
397 118
922 309
20.2 7.9
5, D-Ala4
1100 480
c
1
–
CHO membranes expressing cloned KOP or MOP receptors
were incubated with Dyn A-(1–13) amide or DAMGO concen-
trations, respectively, either alone or in the presence of each
macrocyclic tetrapeptide, as described in Methods. Data pre-
sented are the mean KB values SEM from at least three experi-
ments, except where noted, which were derived from Schild
regression analysis.
aThe analogues exhibited negligible efficacy at KOP and MOP
receptors; <10% relative to Dyn A-(1–13) amide and DAMGO at
KOP and MOP receptors, respectively. bn = 2. cNo antagonist
activity observed at concentrations up to 300 nM (Ross et al.,
2012).
affinities found for analogues 2 and 5 are similar to those
reported previously by Dolle et al. (2009), although those
authors reported substantially lower affinity (Ki > 2 μM) for
peptide 4. None of the analogues showed appreciable affinity
for DOP receptors in the binding assay.
As observed with the parent peptide, none of the Ala
analogues exhibited appreciable agonist activity in the
[35S]GTPγS assay at KOP and MOR receptors. All of the pep-
tides antagonized the stimulation of [35S]GTPγS binding by
Dyn A-(1–13)NH2 at KOP receptors with varying potencies
(Table 2). Analogue 2 exhibited potent antagonist activity at
KOP receptors (KB = 0.8 nM), consistent with the report by
Dolle et al. (2009). The potencies of the other analogues as
KOP receptor antagonists in this assay generally paralleled
their receptor affinities. The potencies of the analogues as
that produced by morphine [ED50 value
5.03) nmol, i.c.v.].
= 2.35 (1.13–
To determine the involvement of MOP, KOP and DOP
receptors in the observed antinociceptive activity, mice were
pretreated with the selective receptor antagonists β-FNA, nor-
BNI and naltrindole, respectively, prior to administration of a
macrocyclic tetrapeptide (Figure 3). Antinociception pro-
duced by analogue 2 was significantly antagonized only by
nor-BNI pretreatment [F(3,27) = 7.26; P = 0.001; one-way ANOVA
with Tukey’s HSD]. In contrast, the antinociception of ana-
logues 3 and 4 were significantly reduced by pretreatment
with selective antagonists for all three opioid receptors [F(3,28)
= 100.0; P < 0.0001 and F(3,27) = 62.7; P < 0.0001 respectively].
The antinociception induced by the D-Ala4 analogue 5 was
3216 British Journal of Pharmacology (2014) 171 3212–3222

Opioid profiles of [D-Trp]CJ-15,208 Ala analogues
BJP
Figure 3
Opioid receptor selectivity of the antinociceptive activity produced
by alanine analogues of [D-Trp]CJ-15,208 in the mouse 55°C warm-
water tail-withdrawal assay in C57BL/6J mice. The antinociceptive
activity of the analogues was determined at the indicated doses after
i.c.v. administration alone (solid bars), 24 h after administration of
β-FNA or nor-BNI, and 15 min after pretreatment with naltrindole.
Tail withdrawal latencies were measured 30 min after analogue
administration. Data shown are mean % antinociception SEM.*P <
0.05, significantly different from response of matching administered
analogue alone; one-way ANOVA followed by Tukey’s post hoc test.
Figure 2
Antinociceptive activity of [D-Trp]CJ-15,208 (1) and the alanine ana-
logues 2–5 in vivo following i.c.v. administration in the 55°C warm-
water tail-withdrawal assay in C57BL/6J mice. Data shown are mean
%
antinociception
SEM. (A) Dose–response curves at peak
response, which was 30 min for 1 or 20 min for analogues 2–5. (B)
Time course for antinociceptive activity for 1 (30 nmol), 2 (10 nmol),
3 (30 nmol), 4 (3 nmol) and 5 (1 nmol).
dependent manner following a 3 h pretreatment [F(4,39) = 13.6;
P < 0.0001 and F(4,39) = 42.0; P < 0.0001, respectively; Figure 5].
The Ala3 analogue
4 proved less potent, antagonizing
significantly antagonized by either β-FNA or nor-BNI pre-
treatment [F(3,28) = 41.0; P < 0.0001], but naltrindole pretreat-
ment was without significant effect.
U50,488 antinociception only after pretreatment with a
higher dose [30 nmol, i.c.v.; F(3,32) = 17.3; P < 0.0001], while
the D-NMePhe2 analogue 3 did not antagonize U50,488 at
doses up to 30 nmol, i.c.v. [F(4,39) = 0.19; P = 0.94]. The dura-
tion of the KOP receptor antagonist activity of the three
active alanine analogues differed (Figure 6). The duration of
the antagonist activity produced by the Ala1 analogue (2,
1 nmol, i.c.v.) was similar to that of the parent macrocyclic
peptide 1 (3 nmol, i.c.v.) (Ross et al., 2012), with significant
KOP receptor antagonism detected for 6–8 h [F(4,39) = 12.8; P <
0.0001; Figure 6]. In contrast, the antagonist activity of ana-
logue 5 (30 nmol, i.c.v.) was substantially reduced by 4 h, and
had completely dissipated by 8 h [F(3,32) = 27.2; P < 0.0001].
The antagonist activity of the Ala3 analogue 4 dissipated even
more rapidly, with significant KOP receptor antagonism
detected after a 3 h pretreatment [F(3,32) = 18.1; P < 0.0001]
that was gone by 4 h (P > 0.05, not significant; Figure 6).
The receptor selectivity of the analogues’ antagonist activ-
ity was determined by further examining the peptides’ ability
to antagonize the MOP receptor-preferring agonist morphine
(10 mg·kg−1, i.p.) and the DOP receptor-selective agonist
SNC-80 (100 nmol, i.c.v., Figure 7). At the same doses
tested for KOP receptor antagonism, none of the alanine
analogues significantly antagonized morphine [F(5,45) = 1.54;
P = 0.20]. Although a globally significant effect was detected
[F(5,46) = 2.68; P = 0.03], none of the individual macrocyclic
The opioid receptor mediation of antinociception
induced by the Ala1 analogue 2 was further assessed in MOP
KO, KOP KO mice and wild-type (WT) mice pretreated with
naltrindole (Figure 4). The antinociceptive activity of this
peptide in MOP KO mice [ED50 = 5.47 (2.02–14.9) nmol, i.c.v.]
or WT mice pretreated with naltrindole [ED50 = 5.47 (2.62–
8.49) nmol, i.c.v.] was not statistically different from the
response observed in naïve WT mice [F(1,65) = 2.07; P = 0.155;
non-linear regression modeling], suggesting little if any con-
tribution of MOP or DOP receptors to the antinociceptive
activity of analogue 2. However, the antinociceptive dose–
response curve for analogue 2 was shifted substantially to the
right in KOP KO mice, with significant antinociception
observed only at doses of 30 nmol or higher (Figure 4). These
data confirmed significant mediation by KOP receptors of the
observed antinociception.
Antagonist activity. The Ala analogues of [D-Trp]CJ-15,208
were evaluated for their ability to antagonize the KOP
receptor-selective agonist U50,488 (10 mg·kg−1, i.p.)
(Figure 5). Consistent with the action of the parent com-
pound 1 (Ross et al., 2012), the Ala1 (2) and D-Ala4 (5)
analogues significantly antagonized U50,488 in
a dose-
British Journal of Pharmacology (2014) 171 3212–3222 3217

J V Aldrich et al.
BJP
Figure 4
Further analysis of opioid receptor mediation of the antinociceptive
activity of analogue 2. The antinociceptive dose–response of ana-
logue 2 in the mouse 55°C warm-water tail-withdrawal assay was
determined in MOP KO mice, KOP KO mice and C57BL/6J WT mice
pretreated 15 min with naltrindole (20 mg·kg−1, i.p.) prior to admin-
istration of 2. Tail withdrawal latencies were measured 20 min after
analogue administration, except in KOP KO mice where the tail
withdrawal latencies were measured 50 min after analogue admin-
istration. Data shown are mean % antinociception SEM.
Figure 6
Duration of macrocyclic tetrapeptide-mediated antagonism of
U50,488-induced antinociception in the mouse 55°C warm-water
tail-withdrawal assay. Mice were pretreated i.c.v. with [D-Trp]CJ-
15,208 [1, 3 nmol; from Ross et al. (2012)] or one of the analogues
displaying KOP receptor antagonism (2, 1 nmol; 4 or 5, 30 nmol
each), and the antinociception induced by U50,488 was determined
3–24 h later. Tail withdrawal latencies were determined 40 min after
U50,488 administration. Data shown are mean % antinociception
SEM. *P < 0.05, significantly different from response of U50,488
administered alone; one-way ANOVA followed by Tukey’s post hoc test.
Figure 7
Figure 5
Opioid receptor selectivity of antagonism by the alanine analogues of
[D-Trp]CJ-15,208 in the mouse 55°C warm-water tail-withdrawal
assay. Antinociception induced by morphine (left group) or SNC-80
(right group) was not significantly decreased by a 3 h pretreatment
with 1 [3 nmol; from Ross et al. (2012)], 2 (10 nmol), 3 (30 nmol), 4
(30 nmol) or 5 (10 nmol), in contrast to the antinociceptive effect of
U50,488 (centre group), which was significantly antagonized by
pretreatment with 1, 2, 4 and 5. Tail withdrawal latencies were
determined 40 min after selective agonist administration. Data
shown are mean % antinociception SEM. *P < 0.05, significantly
different from response of U50,488 administered alone; one-way
ANOVA followed by Tukey’s post hoc test.
Dose-dependent antagonism of U50,488-induced antinociception
by pretreatment with the alanine analogues 2, 4 and 5 compared
with [D-Trp]CJ-15,208 [1; from Ross et al. (2012)] in the mouse 55°C
warm-water tail-withdrawal assay. Mice were pretreated i.c.v. with
the [D-Trp]CJ-15,208 or one of the analogues 2–5, 140 min prior to
administration of the KOP receptor-selective agonist U50,488, and
antinociceptive activity determined 40 min later. Alanine analogue 3
did not antagonize U50,488-induced antinociception at doses up to
30 nmol. Data shown are mean % antinociception SEM. *P < 0.05,
significantly different from response of U50,488 administered alone;
one-way ANOVA followed by Tukey’s post hoc test.
3218 British Journal of Pharmacology (2014) 171 3212–3222

Opioid profiles of [D-Trp]CJ-15,208 Ala analogues
BJP
Figure 8
Time-dependent prevention of reinstatement of extinguished
cocaine–CPP following pretreatment with analogues 2 and 5. (A)
Schematic showing timing of the extinction, treatment and reinstate-
ment protocol. Vehicle (50% DMSO) or analogues 2 or 5 (10 or
30 nmol, i.c.v., respectively) was administered on days 28 and 29,
2 h prior to initial exposure to forced swim stress; for cocaine place
conditioning the mice were treated on days 28 and 29 with vehicle
or peptide, followed by cocaine place conditioning 5 min to 2 h after
the injection (square) on day 29. (B) Mice exhibited significant
preference for the cocaine (10 mg kg−1, s.c. daily for 4 days)-paired
compartment, with extinction occurring over the next 3 weeks (left
bars). Mice were then exposed to forced swim stress (centre bars) or
an additional round of cocaine place conditioning (right bars), result-
ing in the reinstatement of place preference in vehicle-treated mice.
Pretreatment for 2 h with either alanine analogue 2 or 5 prevented
stress-induced, but not cocaine-primed, reinstatement of cocaine-
seeking behaviour in the CPP assay. (C) Analogue 2 also prevented
cocaine-primed reinstatement of cocaine CPP when administered 5
or 30 min, but not 1 or 2 h, prior to cocaine. Pretreatment with
analogue 2 without cocaine (rightmost bar) did not induce reinstate-
ment by itself. Data shown are mean ( SEM) difference in time spent
on the drug-paired side. *P < 0.05, significantly different from pre-
conditioning place preference response (leftmost bar); †P < 0.05,
significantly different from post-CPP response (leftmost solid black
bar); ‡P < 0.05, significantly different from vehicle-treated, stress-
induced or cocaine-primed reinstatement of place preference
response; ANOVA followed by Tukey’s post hoc test.
◀
CPP after exposure to forced swimming [F(5,693) = 35.1; P <
0.0001] or cocaine [F(5,703) = 40.6; P < 0.0001]. Both alanine
analogues 2 and 5 prevented stress-induced reinstatement of
cocaine CPP (P < 0.005; Figure 8B, central bars), but not
cocaine-induced reinstatement (P > 0.05; Figure 8B, right-
most bars), when administered 2 h prior to the forced swim
or administration of cocaine, a time point coinciding with
their KOP receptor antagonist activity. These results are con-
sistent with the action of other KOP receptor-selective
antagonists (Carey et al., 2007; Aldrich et al., 2009), including
the parent peptide 1 (Ross et al., 2012). Because the Ala1
analogue
2 demonstrated short-duration KOP receptor
agonism (<2 h), additional mice were treated with analogue 2
for 5, 30 or 60 min prior to cocaine exposure (Figure 8C).
Mice pretreated with analogue 2 for shorter time periods (5 or
30 min) prior to the additional cycle of cocaine place condi-
tioning demonstrated significant prevention of cocaine-
induced reinstatement [F(8,772) = 25.6; P < 0.0001] (Figure 8C).
Pretreatment of mice demonstrating extinction of cocaine
CPP with the Ala1 analogue 5 min before place preference
testing, but without the additional cocaine conditioning, did
not result in a significant change from the extinction
response (Figure 8C, rightmost bar).
tetrapeptides significantly antagonized SNC-80-induced anti-
nociception (P > 0.05, Tukey’s post hoc test).
Prevention of stress-induced reinstatement of cocaine CPP. We
evaluated the ability of the Ala1 (2) and D-Ala4 (5) analogues
to prevent reinstatement of cocaine CPP (Figure 8). Following
4 days of cocaine place conditioning, mice demonstrated
significant CPP [F(3,827) = 50.6; P < 0.0001] and subsequent
extinction 3 weeks after conditioning (Figure 8B). Mice were
then pretreated daily for two days with either analogue 2
(10 nmol, i.c.v.) or analogue 5 (30 nmol, i.c.v.) and subjected
to forced swimming or an additional round of cocaine con-
ditioning (see schematic, Figure 8A). Mice pretreated with
vehicle demonstrated significant reinstatement of cocaine
Discussion and conclusions
Overall, the in vitro pharmacological results for the alanine-
substituted analogues of [D-Trp]CJ-15,208 were strikingly
similar to those observed for the corresponding substitutions
in the natural product CJ-15,208 containing L-Trp (Aldrich
et al., 2011). In general, alanine substitution had a greater
British Journal of Pharmacology (2014) 171 3212–3222 3219

J V Aldrich et al.
BJP
impact on binding affinities for KOP than for MOP receptors
in both macrocyclic tetrapeptides. Substitution of the Trp
residue in both peptides substantially decreased binding
affinity for both KOP and MOP receptors, suggesting the
importance of the indole moiety for receptor binding, while
substitution of Phe1 with Ala increased affinity for both KOP
and MOP receptors. However, in our studies, the substitution
of Phe3 with Ala appeared to be better tolerated in [D-Trp]CJ-
15,208 than in the L-Trp isomer (Aldrich et al., 2011).
As observed with the alanine analogues of CJ-15,208
(Aldrich et al., 2011), the alanine analogues of the D-Trp
isomer exhibited in vivo pharmacological profiles that were
unexpected based on their opioid receptor affinities and
activity in the [35S]GTPγS assay in vitro. Consistent with the
results for the alanine analogues of CJ-15,208, agonist activity
was not detected in vitro in the GTPγS assay at KOP or MOP
receptors for any of the alanine analogues of 1. While the
parent peptide 1 exhibited significant antinociceptive activity
in the 55°C warm-water tail-withdrawal assay only at an
elevated dose (30 nmol, i.c.v.), alanine substitution for any of
the residues increased antinociceptive potency in this assay
following i.c.v. administration. Interestingly, the relative
potencies of the alanine analogues of 1 in the in vivo assay
were almost completely reversed compared with their relative
affinities for KOP receptors, with the D-Ala4 analogue 5 exhib-
iting the most potent antinociceptive activity in vivo despite
its micromolar affinity for these receptors. Removal of the
indole in analogue 5 resulted in a profile more like the natural
product CJ-15,208, with mixed KOP/ MOP receptor agonism
and KOP receptor antagonist activity.
For most of the analogues, multiple opioid receptors
appear to contribute to their antinociceptive activity. For all
the alanine analogues pretreatment with nor-BNI signifi-
cantly reduced the antinociceptive activity of the analogues,
suggesting KOP receptor mediation of their antinociception.
This is in contrast to the alanine analogues of CJ-15,208
where the antinociceptive activity was mediated predomi-
nantly by MOP receptors (Aldrich et al., 2011). For all of the
analogues except analogue 2, pretreatment with the MOP
receptor antagonist β-FNA significantly decreased antinocic-
eptive activity, suggesting that these receptors also contrib-
uted to their antinociception. Similar to the results for KOP
receptors, the relative antinociceptive potencies of analogues
3, 4 and 5 in vivo did not correlate with their affinities for
MOP receptors. Naltrindole also significantly decreased the
antinociception produced by analogues 3 and 4, suggesting
DOP receptors contributed to their observed antinociceptive
activity. This was unexpected given their very low affinity for
DOP receptors. Together, these results suggest that all of the
amino acid side chains contribute to minimizing the agonist
(antinociceptive) activity in vivo of the parent macrocyclic
peptide 1.
the minimal contribution of MOP receptors to the antinoci-
ception of analogue 2 in spite of its relatively high affinity for
this receptor (Ki = 27 nM).
Each of the alanine analogues of [D-Trp]CJ-15,208 except
the D-NMeAla2 analogue 3 antagonized the antinociceptive
effect of U50,488 in vivo, suggesting that the D-Pro2 residue is
important for KOP receptor antagonist activity in the parent
peptide. The lack of such antagonist activity of analogue 3 in
vivo was unexpected given its KOP receptor antagonist activ-
ity in the GTPγS assay. While the potency of analogue 3 as a
KOP receptor antagonist in vitro was modest, it proved more
potent than analogues 4 and 5, which did exhibit KOP recep-
tor antagonism in vivo. These results contrast with those for
the alanine analogues of CJ-15,208 where all of the analogues
exhibited KOP receptor antagonism in vivo (Aldrich et al.,
2011). Consistent with other peptide KOP receptor antago-
nists (Aldrich et al., 2009; 2011; Ross et al., 2012), the dura-
tion of the antagonist activity of analogues 2, 4 and 5 was
relatively short (<18 h). These results contrast with the estab-
lished non-peptide KOP receptor-selective antagonists nor-
BNI, GNTI and JDTic and their exceptionally long duration of
antagonist activity (weeks after a single dose) (Metcalf and
Coop, 2005). Notably, the very short KOP receptor antago-
nism of analogue 4 (<4 h) suggests it could serve as a useful
pharmacological tool to study KOP receptor-mediated physi-
ological and pharmacological activities.
While these analogues produce antinociception and,
except for analogue 3, antagonist activity that is clearly medi-
ated through opioid receptors, the differences between the in
vitro and in vivo activity profiles suggest that these com-
pounds produce their opioid activity through more complex
mechanisms than utilized by typical opioid receptor ligands.
As discussed above, similar differences were noted for the
alanine analogues of the natural product CJ-15,208 (Aldrich
et al., 2011), and have also been found for other novel anti-
nociceptive compounds that are structurally unrelated to
these macrocyclic peptides (Reilley et al., 2010). There are a
number of unconventional mechanisms that could poten-
tially account for the differences observed between the in vitro
and in vivo assays, including activation of different signalling
pathways, modulation of opioid receptors by other proteins
in vivo (including other receptors) and modulation of endog-
enous opioid peptide levels (Szeto et al., 2003). We are very
interested in understanding the mechanism(s) behind the
complex in vivo activity profiles of these compounds that
could offer important new insights into both opioid function
and approaches to drug discovery. These investigations,
however, will involve extensive additional studies that are
beyond the scope of this report describing the basic charac-
terization of these unusual opioid compounds. Nevertheless,
the observed differences highlight the important contribu-
tions that in vivo studies can make to understanding the
pharmacological activity profiles of novel ligands.
The KOP receptor mediation of the antinociceptive activ-
ity of the Ala1 analogue 2 was confirmed in the KOP KO mice,
which showed a significant rightward shift in the dose–
response curve compared with WT mice (Figure 4). The
lowered maximal response in the KOP KO mice suggests
analogue 2 also produces partial agonism through the other
opioid receptors, but only at higher doses. The antinocicep-
tive potency of analogue 2 was not significantly different in
MOP KO mice compared with WT mice, further supporting
Because of their KOP receptor antagonist activity, alanine
analogues 2 and 5 were examined for their ability to prevent
both stress- and cocaine-induced reinstatement of cocaine
CPP. Consistent with the action of other KOP receptor-
selective antagonists (Carey et al., 2007; Aldrich et al., 2009;
Ross et al., 2012), pretreatment with either peptide at a time
point when they exhibited KOP receptor antagonist activity
(2 h) prevented stress-induced, but not cocaine-primed,
3220 British Journal of Pharmacology (2014) 171 3212–3222

Opioid profiles of [D-Trp]CJ-15,208 Ala analogues
BJP
reinstatement of cocaine CPP. When evaluated for its ability
to prevent cocaine-primed reinstatement following shorter
pretreatment times, the Ala1 analogue 2 prevented reinstate-
ment with a time course consistent with its antinociceptive
activity. Evaluation in KOP KO mice (Figure 4) verified that at
this dose (10 nmol) used in the CPP studies the agonist activ-
ity of 2 was solely due to activation of KOP receptors. The
ability of analogue 2 to prevent both stress- and cocaine-
induced reinstatement of extinguished cocaine-seeking
behaviour is an unusual property. This sets this peptide apart
from typical single-action KOP receptor ligands; compounds
that produce only KOP receptor antagonism do not prevent
cocaine-primed reinstatement (Beardsley et al., 2005; Carey
et al., 2007; Aldrich et al., 2009), and repeated treatment with
KOP receptor-selective agonists may reinstate cocaine-seeking
behaviour (Redila and Chavkin, 2008). The results with ana-
logue 2 are consistent with those found for the mixed
agonist/ KOP receptor antagonist CJ-15,208, which also pre-
vented cocaine-induced reinstatement following a short pre-
treatment time (Aldrich et al., 2013). With both KOP receptor
agonist and antagonist activities, these macrocyclic peptides
can counteract both stress- and drug-induced triggers of rein-
statement in a time-dependent manner, suggesting com-
pounds with broader therapeutic value in maintaining
abstinence in cocaine abusers.
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Supporting information
Additional Supporting Information may be found in the
online version of this article at the publisher’s web-site:
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Houghten RA (2010). Identification of two novel, potent,
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http://dx.doi.org/10.1111/bph.12664
Appendix S1 Analytical data for compounds 2-5.
3222 British Journal of Pharmacology (2014) 171 3212–3222