The macrocyclic peptide natural product CJ-15,208 is orally active and prevents reinstatement of extinguished cocaine-seeking behavior

Article abstract of DOI:10.1021/np300697k

The macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-d-Pro- Phe-Trp]) exhibited both dose-dependent antinociception and kappa opioid receptor (KOR) antagonist activity after oral administration. CJ-15,208 antagonized a centrally administered KOR selective agonist, providing strong evidence it crosses the blood-brain barrier to reach KOR in the CNS. Orally administered CJ-15,208 also prevented both cocaine- and stress-induced reinstatement of extinguished cocaine-seeking behavior in the conditioned place preference assay in a time- and dose-dependent manner. Thus, CJ-15,208 is a promising lead compound with a unique activity profile for potential development, particularly as a therapeutic to prevent relapse to drug-seeking behavior in abstinent subjects.

Full text of DOI:10.1021/np300697k

Article
pubs.acs.org/jnp
The Macrocyclic Peptide Natural Product CJ-15,208 Is Orally Active
and Prevents Reinstatement of Extinguished Cocaine-Seeking
Behavior
Jane V. Aldrich,*,† Sanjeewa N. Senadheera,† Nicolette C. Ross,‡,§ Michelle L. Ganno,‡ Shainnel O. Eans,‡
and Jay P. McLaughlin‡
†Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, United States
‡Torrey Pines Institute for Molecular Studies, Port St. Lucie, Florida 34987, United States
ABSTRACT: The macrocyclic tetrapeptide natural product CJ-15,208
(cyclo[Phe-D-Pro-Phe-Trp]) exhibited both dose-dependent antinociception
and kappa opioid receptor (KOR) antagonist activity after oral administration.
CJ-15,208 antagonized a centrally administered KOR selective agonist,
providing strong evidence it crosses the blood−brain barrier to reach KOR
in the CNS. Orally administered CJ-15,208 also prevented both cocaine- and
stress-induced reinstatement of extinguished cocaine-seeking behavior in the
conditioned place preference assay in a time- and dose-dependent manner.
Thus, CJ-15,208 is a promising lead compound with a unique activity profile
for potential development, particularly as a therapeutic to prevent relapse to
drug-seeking behavior in abstinent subjects.
he tetrapeptide CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]),
which was isolated from the fermentation broth of the
withdrawal assay mediated by KOR and mu opioid receptors
(MOR), followed by KOR antagonist activity at later time
points.5 The D-Trp peptide also binds preferentially to KOR
with similar affinity to that of the natural product,3−6 but in
contrast to the L-Trp peptide exhibits primarily KOR antagonist
activity in vivo.5 Other macrocyclic peptides have also
demonstrated opioid activity. Structurally related macrocyclic
pentapeptides (cyclo[Tyr-X-D-Trp-Phe-Y]), designed as cyclic
analogues of the linear MOR endogenous agonist endomorphin
1, have been reported with affinity and efficacy at MOR7,8 and
recently with affinity for KOR.8
T
fungus Ctenomyces serratus ATCC 15502, was reported to bind
preferentially to kappa opioid receptors (KOR) and to
antagonize a KOR agonist in the electrically stimulated rabbit
vas deferens assay.1 This macrocyclic peptide is structurally
distinct from endogenous opioid peptides, as it lacks the classic
tyrosine pharmacophore containing a basic amine and also the
additional basic groups found in the C-terminal “address”
sequence2 of the dynorphin endogenous KOR agonists.
The physiochemical properties of CJ-15,208, specifically its
macrocyclic structure and relatively low molecular weight (577
Da), suggested it would be a promising lead compound
expected to exhibit activity after oral administration. In contrast
to linear peptides, which are generally metabolized rapidly by
proteases, macrocyclic peptides are typically stable to proteases
and instead can be metabolized by cytochrome P450 enzymes
involved in drug metabolism.9,10 Their macrocyclic structure
also often promotes intramolecular hydrogen bonding, which
can facilitate membrane permeability,11 thereby enhancing oral
bioavailability and penetration of the blood−brain barrier into
the central nervous system (CNS). The immunosuppressant
drug cyclosporine is active after oral administration, providing
precedent for the oral activity of macrocyclic peptides.
Thus CJ-15,208 provided a novel lead compound for the
development of new ligands for KOR. When the natural
product was originally isolated, the stereochemistry of three of
the residues (D-Pro and the two Phe residues) was determined,
but not that of the tryptophan residue.1 We synthesized the
peptides containing L- and D-Trp3,4 and found that the optical
rotation of the peptide containing L-Trp was consistent with
that of the natural product. However, when this L-Trp-
containing macrocyclic peptide was tested in vivo following
intracerebroventricular (icv) injection, it exhibited potent
antinociceptive activity in the mouse 55 °C warm-water tail-
Building on our previous results,5 CJ-15,208 was evaluated in
mice after oral administration in three assays: the 55 °C warm-
Special Issue: Special Issue in Honor of Lester A. Mitscher
Received: October 5, 2012
Published: January 17, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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water tail-withdrawal assay for antinociceptive and KOR
antagonist activity, the rotorod assay for locomotor effects,
and a conditioned place preference (CPP) assay for its ability to
block reinstatement of extinguished cocaine-seeking behavior.
Herein it is reported that the oral administration of CJ-15,208
produces dose-dependent antinociception and KOR antagonist
activity and prevents reinstatement of cocaine-seeking behavior
induced by exposure to either stress or cocaine.
more prolonged antinociceptive activity (>140 min), which was
significantly greater than the 10 or 30 mg/kg doses (F(2,29)
11.89, p < 0.05; one-way ANOVA).
=
The ability of CJ-15,208 to antagonize the antinociceptive
activity of the KOR-selective agonist U50,488 (( )-trans-3,4-
dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-
benzeneacetamide) was then assessed following oral admin-
istration of the macrocyclic peptide (Figure 2). As expected, CJ-
RESULTS AND DISCUSSION
■
In order to obtain sufficient macrocyclic peptide for evaluation
after oral administration, the synthesis and purification of CJ-
15,208 was modified12 with changes made in the key cyclization
reaction and purification procedure. The linear peptide Trp-
Phe-D-Pro-Phe was cyclized at 30 °C for 24 h after 12 h at
room temperature. Purification by normal-phase flash chroma-
tography rather than reversed-phase HPLC12 greatly facilitated
purification of the larger quantities of the peptide required for
evaluation in vivo following oral administration; with these two
modifications >400 mg of pure macrocyclic peptide (75%
yield) was obtained.
The macrocyclic peptide was initially evaluated in vivo in the
55 °C warm-water tail-withdrawal antinociceptive assay to
assess its agonist and antagonist activity after oral admin-
istration. CJ-15,208 exhibited antinociceptive activity with an
ED50 value (and 95% confidence interval) of 3.49 (1.98−5.73)
mg/kg, po (per os) 40 min after administration (Figure 1),
Figure 2. Oral administration of CJ-15,208 produced dose-dependent
antagonism of U50,488-induced antinociception administered (A)
peripherally (ip) and (B) centrally (icv) in the mouse 55 °C warm-
water tail-withdrawal assay. Mice were pretreated with CJ-15,208 3 h
prior to antinociceptive testing with U50,488. The mean %
antinociception
SEM from 8 mice per group is presented; * =
significantly different from the baseline tail-withdrawal latency; † =
significantly different from antinociception induced by U50,488 alone;
one-way ANOVA with Tukey HSD post hoc test.
15,208 antagonized peripherally administered U50,488 [10 mg/
kg, through the intraperitoneal (ip) route] in a dose-dependent
fashion (Figure 2A), with significant antagonism at doses of
both 3 and 10 mg/kg, po (F(3,51) = 64.41, p < 0.0001; one-way
ANOVA). To assess whether CJ-15,208 could penetrate the
blood−brain barrier to reach KOR in the CNS, the ability of
the macrocyclic peptide to antagonize centrally administered
U50,488 was then evaluated. Oral administration of CJ-15,208
3 h prior to analgesic testing dose-dependently antagonized the
antinociceptive effect of icv U50,488 (30 nmol, Figure 2B) at
doses of 30 and 60 mg/kg, po (F(4,63) = 139.7, p < 0.0001; one-
way ANOVA), providing strong evidence that this macrocyclic
peptide can cross the blood−brain barrier.
KOR-selective agonists are known to produce sedative effects
through activation of KOR.13 To assess whether the KOR
agonist activity of CJ-15,2085 would produce sedative effects
that could confound analysis in the antinociceptive or
conditioned place preference assays (see below), the macro-
cyclic peptide was evaluated in the rotorod assay (Figure 3). As
expected, the KOR-selective agonist U50,488 (10 mg/kg, ip)
Figure 1. Antinociceptive activity of CJ-15,208 following oral
administration to C57BL/6J mice using the 55 °C warm-water tail-
withdrawal assay with repeated measurement over time. Admin-
istration of vehicle (1:1:8) alone had no significant effect. The mean %
antinociception SEM from 7 to 24 mice per group is presented.
consistent with its demonstrated opioid agonist activity after icv
administration.5 Over the first hour, CJ-15,208 exhibited a
significant interaction of dose and time (F(36,480) = 4.25, p <
0.05; repeated measures analysis of variance, ANOVA),
reaching peak antinociception within 40 min that did not
significantly decline by 60 min. Neither vehicle nor the 0.3 mg/
kg dose was efficacious during this time. Over the second hour,
CJ-15,208 again demonstrated significant interactions of dose
and time (F(25,250) = 5.35, p < 0.05; repeated measures
ANOVA), but with reductions in antinociception such that by
120 min, post-treatment tail-withdrawal latencies after 1, 3, 10,
or 30 mg/kg, po CJ-15,208 did not significantly differ from the
vehicle. Notably, the highest dose (60 mg/kg, po) produced
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Figure 3. CJ-15,208 was without locomotor effect in the mouse
rotorod assay. Mice were orally administered vehicle, CJ-15,208 (10 or
60 mg/kg, po), or U50,488 (10 mg/kg, ip). Latency to fall SEM is
presented as the mean percent change from baseline performance for 7
or 8 mice per treatment group; * = significantly different from
matching vehicle response.
caused a significant reduction (F(4,232) = 30.55, p < 0.0001; two-
way ANOVA) in the latency to fall from the rotating rod
compared to vehicle at several time points, consistent with the
sedative effects of centrally active KOR agonists. In contrast,
mice treated with CJ-15,208 at 10 or 60 mg/kg, po did not
exhibit significant differences in the latency to fall compared to
vehicle-treated animals. Thus, orally administered CJ-15,208
does not appear to produce the sedation typically seen with
KOR-selective agonists such as U50,488.
Figure 4. Dose-dependent prevention of reinstatement of cocaine
CPP following oral pretreatment with CJ-15,208. (A) Schematic of
reinstatement and testing protocol. Vehicle (1:1:8, po) or CJ-15,208
(30 or 60 mg/kg, po) was administered on days 28 and 29, 3 h prior to
exposure to forced swim stress (diamonds). For cocaine-primed
reinstatement the mice were also treated on days 28 and 29 with
vehicle or CJ-15,208, followed by cocaine place conditioning 30 min
later on day 29 (square). (B) Mice exhibited significant preference for
the cocaine (10 mg/kg, sc, daily for 4 days)-paired compartment, with
extinction occurring 3 weeks later (left bars). Mice were then exposed
to forced swim stress (center bars) or an additional round of cocaine
place conditioning (right bars), resulting in the reinstatement of place
preference in vehicle-treated mice (hashed bars). Pretreatment with
CJ-15,208 (middle bars) 3 h prior to forced swimming prevented
stress-induced reinstatement of place preference, and pretreatment 30
min prior to cocaine conditioning (right bars) prevented cocaine-
primed reinstatement of cocaine CPP. Mean difference in time spent
on the drug-paired side SEM is presented for 12 to 16 mice per
treatment condition; cocaine place conditioning data on left represent
combined responses of 96 mice used in this experiment; * =
significantly different from preconditioning place preference response
(leftmost bar), † = significantly different from post-CPP response
(leftmost black solid bar); ‡ = significantly different from stress-
induced or cocaine-primed reinstatement of place preference response
(center and right); ANOVA followed by Tukey HSD post hoc test.
KOR and its endogenous agonists the dynorphins play an
important role in the response to stress14−16 and are thought to
mediate reinstatement of cocaine-seeking behavior following
extinction.17,18 A number of KOR antagonists have been shown
to prevent reinstatement of extinguished cocaine-seeking
behavior,18−22 including the structurally related KOR antago-
nist [D-Trp]CJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp]).5 Since
CJ-15,208 produced KOR antagonist activity, the ability of oral
pretreatment with CJ-15,208 to prevent stress-induced
reinstatement of extinguished cocaine CPP was evaluated.
Following four days of cocaine place conditioning, mice showed
a significant preference for the cocaine-paired chamber (F(10,467)
= 11.34; p < 0.0001; one-way ANOVA with Tukey honestly
significant difference (HSD) post hoc test; Figure 4, black bar).
Extinction was observed within 3 weeks of place conditioning,
at which point mice were pretreated orally once daily for two
days with vehicle (1:1:8) or CJ-15,208 (30 or 60 mg/kg, po) 3
h before exposure to forced swimming stress (see diamonds in
schematic, Figure 4A). The duration of pretreatment was
chosen to correspond to CJ-15,208-mediated KOR antagonist
activity in the CNS (see Figure 2B). Consistent with its KOR
antagonism, CJ-15,208 prevented stress-induced reinstatement
of cocaine CPP at 60 (p < 0.05, Tukey HSD post hoc test) but
not 30 mg/kg, po (Figure 4B).
antinociceptive assay (Figure 1). Mice demonstrating extinction
of cocaine CPP were pretreated orally once daily for two days
with vehicle (1:1:8) or CJ-15,208 (30 or 60 mg/kg, po) and
exposed to an additional cycle of cocaine place conditioning on
day 29 (see square in schematic, Figure 4A). When
administered orally 30 min prior to cocaine, CJ-15,208
prevented cocaine-induced reinstatement of cocaine CPP (p
< 0.05, Tukey HSD post hoc test, only after pretreatment with
60 mg/kg, po). Notably, pretreatment with CJ-15,208 (60 mg/
kg/d, po, for 2 d) did not induce reinstatement in mice
demonstrating extinction of cocaine CPP where saline was
substituted for the additional cycle of cocaine place
KOR agonists block acute cocaine self-administration,
suppress the rewarding effects of cocaine in animal
models,23−28 and are not themselves reinforcing.29,30 Acute
administration of KOR agonists has also been shown to prevent
the cocaine-primed reinstatement of extinguished cocaine-
seeking behavior.31−33 Since CJ-15,208 initially demonstrated
finite KOR agonist activity, it was examined for its ability to
prevent cocaine-induced reinstatement of cocaine CPP at a
time point (30 min) when it exhibited agonist activity in the
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peptide (300 mg, 0.50 mmol) in DMF (20 mL) was added dropwise at
a rate of 1.2 mL/h, as described above. The reaction was then stirred
for 12 h at room temperature, followed by an additional 24 h at 30 °C.
The solvent was evaporated under reduced pressure at 37 °C, and the
crude macrocyclic tetrapeptide was redissolved in toluene (3 × 25 mL)
and evaporated to dryness. The resulting residue was dissolved in
EtOAc (100 mL), and the organic phase washed sequentially with 1 N
citric acid, saturated NaHCO3 solution, and brine (3× each). The
organic phase was dried over anhydrous MgSO4, and the filtrate
evaporated under reduced pressure to give the crude macrocyclic
tetrapeptide, which was purified by silica gel chromatography using a
step gradient solvent system [EtOAc−hexane (3:2, 100 mL), EtOAc−
hexane (7:3, 100 mL), EtOAc−hexane (4:1, 100 mL), EtOAc−hexane
(9:1, 200 mL), EtOAc (100%, 200 mL), and 1% MeOH in EtOAc
(200 mL)] to afford pure cyclo[Phe-D-Pro-Phe-Trp], which was then
lyophilized from aqueous MeCN to give the peptide as a white solid
(436 mg, 75% yield): 1H NMR (500 MHz, CD3COCD3) δ 9.92 (1H,
s, NH-Trp), 7.66 (1H, d, J = 6.8 Hz, aryl CH/NH), 7.60 (1H, d, J =
10.2 Hz, aryl CH/NH), 7.52 (1H, d, J = 7.9 Hz, aryl CH/NH), 7.38−
7.20 (10H, m, aryl CH/NH), 7.10−7.04 (2H, m, aryl CH), 7.03−6.96
(2H, m, aryl CH), 6.84 (1H, d, J = 1.6 Hz, aryl CH/NH), 5.15−5.13
(1H, m, Phe1 α H), 4.72 (1H, d, J = 6.6 Hz, Pro α H), 4.60−4.55 (1H,
m, Phe3 α H), 3.99−3.95 (1H, m, Trp α H), 3.77−3.71 (1H, m, Pro δ
H), 3.57 (1H, dd, J = 14.7, 8.8 Hz, Trp β H), 3.50 (1H, dd, J = 14.7,
7.9 Hz, Trp β H), 3.34−3.26 (1H, m, Pro δ H), 3.22 (1H, dd, J = 13.6,
8.4 Hz, Phe1 β H), 3.15 (1H, dd, J = 13.7, 9.0 Hz, Phe3 β H), 3.02
(1H, dd, J = 13.6, 7.2 Hz, Phe1 β H), 2.91−2.86 (1H, m, Phe3 β H),
2.29−2.20 (1H, m, Pro β H), 2.02−1.96 (1H, m, Pro γ H), 1.80−1.74
(1H, m, Pro β H), 1.70−1.63 (1H, m, Pro γ H); 13C NMR (125 MHz,
CD3COCD3) δ 175.0 (CO, Trp), 174.4 (CO, Phe3), 173.9 (CO,
Phe1), 172.0 (CO, Pro), 138.6 (ipso C, Phe3), 138.4 (ipso C, Phe1),
137.6 (C, Trp-bridge carbon between 1 and 7), 130.2 (CH, Phe3
meta), 130.1 (CH, Phe1 meta), 129.4 (CH, Phe3 ortho), 129.3 (CH,
Phe1 ortho), 128.2 (C, Trp-bridge carbon between ipso C and 4), 127.5
(CH, Phe1 para and Phe3 para), 124.4 (CH, Trp-2), 122.2 (CH, Trp-
6), 119.6 (CH, Trp-5), 119.0 (CH, Trp-4), 112.3 (CH, Trp-7), 111.2
(ipso C, Trp), 61.7 (CH, Trp α), 58.4 (CH, Pro α), 56.8 (CH, Phe3
α), 54.0 (CH, Phe1 α), 47.1 (CH2, Pro δ) 37.0 (CH2, Phe3 β), 36.9
(CH2, Phe1 β), 26.3 (CH2, Trp β), 25.5 (CH2, Pro β), 25.3 (CH2, Pro
γ); MS (ESI (+)) m/z calcd for [C34H35N4O5 + Na]+ 600.2587, found
600.2560; HPLC tR = 12.6 min (20−85% MeCN over 45 min with
0.1% TFA) and tR = 18.4 min (30−70% MeOH over 45 min with 0.1%
TFA), flow rate 1.0 mL/min, purity >99% in both HPLC systems.
Animals and Compound Administration. Adult male C57BL/
6J mice weighing 20−25 g, obtained from Jackson Laboratories (Bar
Harbor, ME, USA), were used for these studies because of their
established responses to analgesics,42 stress, and cocaine place
conditioning.14,20,21 All mice were kept on a 12 h light−dark cycle
and were housed in accordance with the National Institutes of Health
Guide for Care and Use of Laboratory Animals. CJ-15,208 was dissolved
daily prior to administration in ethanol and Tween 80, and warm (40
°C) saline added to give a final vehicle consisting of 1 part ethanol, 1
part Tween 80, and 8 parts sterile saline (0.9%), referred to as 1:1:8.
This vehicle has been used to solubilize other hydrophobic opiates for
in vivo studies.43,44 U50,488 was administered in 0.9% sterile saline.
Antinociceptive Assay. The 55 °C warm-water tail-withdrawal
assay was performed in mice as previously described,5 with the latency
of tail withdrawal from the water taken as the end point. After
determining baseline tail-withdrawal latencies, mice were administered
CJ-15,208 po in the 1:1:8 vehicle. A cutoff time of 15 s was used in this
study; if the mouse failed to display a tail-withdrawal response during
that time, the tail was removed from the water and the animal was
assigned a maximal antinociceptive score of 100%. At each time point,
antinociception was calculated according to the formula:
conditioning (final preference = −304 71 s, n = 8; p > 0.05,
not significant; Tukey HSD post hoc test).
The ability of CJ-15,208 to prevent both stress- and cocaine-
induced reinstatement of extinguished cocaine-seeking behavior
is an unusual property of this macrocyclic peptide; to our
knowledge this is the first report of an opioid ligand that can
block both of these causes of reinstatement to cocaine-seeking
behavior. Compounds possessing only KOR antagonist activity
have not been shown to prevent cocaine-primed reinstatement
of cocaine-seeking behavior.5,19−21 Reports suggest that KOR-
selective agonists produce dysphoria34 and can actually
reinstate extinguished cocaine-seeking behavior.17,18 It seems
likely that the unique profile of CJ-15,208 in preventing both
triggers of reinstatement is due to the distinctive mixed KOR/
MOR agonist and KOR antagonist activity of this natural
product. Mixed-action KOR/MOR agonists such as the
benzomorphans ethylketocyclazocine and 8-carboxamidocycla-
zocine acutely decrease cocaine self-administration.35,36 Like-
wise, buprenorphine, a mixed MOR partial agonist/KOR
antagonist, has been shown to reduce cocaine self-admin-
istration37,38 and drug (but not stress)-induced reinstatement.39
In conclusion, CJ-15,208 demonstrated both agonist
(antinociception) and KOR antagonist activity following oral
administration. Such a demonstration of biological activity by a
peptide after oral administration is relatively rare.40 The ability
of orally administered CJ-15,208 to antagonize centrally
administered KOR agonist provides strong evidence that this
macrocyclic peptide crosses the blood−brain barrier to reach
KOR in the CNS. In contrast to U50,488, CJ-15,208 does not
decrease the latency to fall in the rotorod test, suggesting that it
is not producing hypolocomotor effects. At pretreatment times
correlating with agonist (antinociceptive) and KOR antagonist
activity in the antinociceptive assay, orally administered CJ-
15,208 prevented cocaine- and stress-induced reinstatement,
respectively, of extinguished cocaine-seeking behavior in the
CPP assay. These results suggest this macrocyclic peptide
natural product is a promising lead compound for potential
treatment of drug abuse, especially for preventing relapse.
EXPERIMENTAL SECTION
■
Materials. Reagents for peptide synthesis were obtained from the
following sources: Fmoc (fluorenylmethoxycarbonyl)-protected amino
acids, 2-chlorotrityl chloride resin, and coupling reagents HATU (2-
(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate) and PyBOP (benzotriazol-1-yloxytripyrrolidinophospho-
nium hexafluorophosphate) were obtained from Novabiochem
(EMD), San Diego, CA, USA; 1-hydroxybenzotriazole and N,N-
diisopropylethylamine were from Fluka, Milwaukee, WI, USA;
trifluoroacetic acid was purchased from Pierce, Rockford, IL, USA;
and HPLC-grade solvents were from Fisher Scientific, Pittsburgh, PA,
USA. HPLC analysis was performed on a Vydac 218TP C18 reversed-
phase column (Grace Davison, 4.6 × 50 mm, 5 μm). Compounds
other than CJ-15,208 used in pharmacological assays were obtained
from Sigma, St. Louis, MO, USA.
Synthesis of CJ-15,208. The linear peptide precursor Trp-Phe-D-
Pro-Phe was synthesized by Fmoc solid-phase synthesis on the 2-
chlorotrityl chloride resin by procedures described previously.3,4,41 The
crude linear peptide was cyclized by a modification of a previously
published procedure.12 The crude linear peptide (300 mg, 0.50 mmol)
in N,N-dimethylformamide (DMF, 20 mL) was added dropwise at a
rate of 1.2 mL/h (using a KD Scientific single infusion syringe pump)
to a dilute solution of HATU (288 mg, 0.75 mmol, 1.5 equiv) and
N,N-diisopropylethylamine (700 μL, 4.0 mmol, 8 equiv) in DMF (800
mL) over 6 h. After 6 h, additional HATU (288 mg, 0.75 mmol, 1.5
equiv) was added to the reaction in one portion, and additional linear
%antinociception = 100 × [( test latency − control latency)
/(15 − control latency )]
For evaluation of its antagonist activity CJ-15,208 was administered
orally, followed 3 h later (when CJ-15,208-induced agonist activity had
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dissipated) by either an ip (10 mg/kg) or icv (30 nmol) injection of
U50,488. The mice were then evaluated 40 and 20 min later,
respectively, in the 55 °C warm-water tail-withdrawal assay as
described above.
Present Address
§Scripps Research Institute, Jupiter, Florida, United States.
Notes
The authors declare no competing financial interest.
Rotorod Assay. Both possible sedative and hyperlocomotor effects
of CJ-15,208 were assessed by rotorod performance, as modified from
previous protocols.45 Following seven habituation trials (the last
utilized as a baseline measure of rotorod performance), mice were
administered vehicle (1:1:8) or CJ-15,208 (10 or 60 mg/kg, po) orally
or U50,488 (10 mg/kg, ip) and assessed in accelerated speed trials
(180 s maximum latency at 0−20 rpm) over a 60 min period.
Decreased latencies to fall in the rotorod test indicate impaired motor
performance. Vehicle-treated animals exhibited significantly increased
latencies to fall over time in this assay (F(6,183) = 3.562, p = 0.0023;
two-way ANOVA with Tukey HSD post hoc test; Figure 3), consistent
with improved mastery of the task over time. Data are expressed as the
percent change from baseline performance.
ACKNOWLEDGMENTS
■
This work was supported by NIDA grants DA023924 and
DA032928 (to J.V.A.) and the State of Florida, Executive Office
of the Governor’s Office of Tourism, Trade, and Economic
Development (to J.P.M.).
DEDICATION
■
Dedicated to Dr. Lester A. Mitscher, of the University of
Kansas, for his pioneering work on the discovery of bioactive
natural products and their derivatives.
Reinstatement of Cocaine Conditioned Place Preference.
Mice were conditioned following a counterbalanced cocaine CPP
paradigm using similar timing to that reported previously.20,21,45 The
amount of time mice spent in each of three compartments was
measured over a 30 min testing period; prior to conditioning animals
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AUTHOR INFORMATION
■
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Corresponding Author
*Tel: (785) 864-2287. Fax: (785) 864-5326. E-mail: jaldrich@
ku.edu.
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