680 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5
Barlocco et al.
diphenhydramine (hydrochloride) from De Angeli (Milan,
Italy). (()-Epibatidine was obtained from Research Biochemi-
cals International (Natick, MA). (-)-Nicotine (hydrogen tar-
trate salt) and mecamylamine (hydrochloride) were purchased
from Sigma Chemical Co. (St. Louis, MO). [3H]-(-)-Cytisine
was obtained from NEN Life Sciences (Boston, MA).
In Vivo Exp er im en ts. Analgesic activity was evaluated
in male Swiss-Webster mice (22-28 g; Morini, San Polo
d’Enza, Italy). Mice were kept at 23 ( 1 °C, with a 12-h light/
dark cycle, light at 7 a.m., with food and water ad libitum. All
experiments were carried out according to the guidelines of
the European Community Council on animal care.
HEK-293 cells (ATCC, Rockville, MD) stably expressing the
human R4â2 nAChR subunit combination, K177 cells, were
maintained as previously described.22 TE671 cells (ATCC,
Rockville, MD) were grown according to the protocol of Lukas.23
Cells were plated out at a density of 1 × 10-6 cells/well on
black-walled, clear-bottomed 96-well plates and used ap-
proximately 72 h after plating. All plates were coated with
poly(ethylenimine) to aid in the adherence of the cells to the
plate.
4. n ACh R F u n ction a l Activity: Changes in the intrac-
ellular free Ca2+ concentrations were measured using the
calcium-chelating dye Fluo-3 (Molecular Probes, Eugene, OR)
in conjunction with a FLIPR (Molecular Devices, Sunnyvale,
CA). The cell permeant acetoxymethyl (AM) ester form of
Fluo-3 was prepared to a concentration of 1 mM in anhydrous
DMSO and 10% pluronic acid. The dye was then diluted to a
final concentration of 4 mM in growth media and placed on
the cells for 1 h at 37 °C. Black-walled 96-well plates were
utilized to reduce light scattering. The unincorporated dye
was removed from the cells by excessive washing with Hepes-
salts buffer (composition, mM: Hepes, 20; NaCl, 120; KCl, 5;
MgCl2, 1; glucose, 5; and CaCl2, 5; pH 7.4 at 25 °C). After
addition of various concentrations of agonists, the Ca2+
dynamics were observed in the FLIPR apparatus equipped
with an argon laser (wavelength, 480 nm), an automated 96-
channel pipettor, and a CCD camera. The intensity of the
fluorescence was captured by the CCD camera every second
for the first minute following the agonist addition with
additional readings every 5 s for a total time period of 5 min.
These images were digitally transferred to an interfaced PC
and changes in fluorescence intensity processed for each
well. The exposure setting of the camera was 0.4 s with f
stop setting of 2 µm. The percent maximal intensity relative
to that induced by 100 µM nicotine was plotted against
the concentration of agonist, and EC50 values were calcu-
lated. Independent measurements of 100 µM nicotine (100%)
and unloaded cells (0%) were performed on each plate of
cells with an average range of 20 000 fluorescence units.
These controls allowed for normalization across several plates
of cells.
Hot plate method described by O’Callaghan and Holtzman15
was used to assess the potential analgesic activity of the test
compounds. The plate temperature was fixed at 52.5 ( 0.1
°C. Mice with a licking latency below 12 and over 18 s in the
test before drug administration (30%) were rejected. An
arbitrary cutoff time of 45 s was adopted. The number of mice
treated in each test varied from 5 to 12. To evaluate the
analgesic efficacy of the new products, their level of analgesia
reached after the injection of the maximal dose unable to
modify mouse rota-rod performance was compared to that of
morphine (8 mg/kg sc) or epibatidine (5 µg/kg sc), taken as
reference compounds. The integrity of motor coordination was
assessed on the basis of the endurance time of the animals on
the rotating rod, according to Vaugh.16 Analgesic efficacy of
each compound (X) is expressed as percentage of that of
epibatidine (5 µg/kg sc) or morphine (8 mg/kg sc). The
calculation was performed using the following formula:
% of analgesic efficacy of X ) (T1 - T2)/(T3 - T4) × 0.100
where T1 is maximum reaction time of X, T2 is pretest reaction
time of X, T3 is maximum reaction time of morphine or
epibatidine, and T4 is pretest reaction time of morphine or
epibatidine.
The maximal nontoxic dose is considered the highest dose
of X which does not cause any visible change in animal
behavior such as tremors, convulsions, hypomotility, etc., so
that the researchers were unable to distinguish between
treated and untreated mice. The evaluation of the behavioral
parameters was performed according to Irwing17 on a group
of at least 10 animals. The abdominal constriction test was
performed according to Koster et al.18 Standard errors on the
values expressed as percentage were not evaluated. Original
data, however, have been statistically processed by employing
Dunnett’s two-tailed test in order to verify the significance of
the differences between the means shown by treated mice at
the maximum reaction time and the pretest reaction time.
Differences were considered statistically significant when P
< 0.05. Percent values were calculated only for those differ-
ences that were statistically significant. In other case, drugs
were considered inactive. Intracerebroventricular (icv) ad-
ministration was performed under ether anesthesia using
isotonic saline as a solvent, according to the method described
by Haley and McCormick.19
Ack n ow led gm en t. The authors are deeply indebted
to Dr. Michael Williams and Dr. Michael Meyer (Abbott
Laboratories) for their support of the in vitro evaluation
of the compounds. Financial support by the Ministero
dell’Universita` e della Ricerca Scientifica e Tecnologica
(Rome) is also gratefully acknowledged.
1
Su p p or tin g In for m a tion Ava ila ble: Tables of H NMR
data, effects of 1 and 2 in the rota-rod test, and results of
modeling (3 pages). See any current masthead page for
ordering information.
Refer en ces
(1) Spande, T. F.; Garraffo, M.; Edwards, M. W.; Yeh, J . C.; Pannell,
L.; Daly, J . W. Epibatidine: A novel (chloropyridyl)azabicyclo-
heptane with potent analgesic activity from ecuadoran poison
frog. J . Am. Chem. Soc. 1992, 114, 3475-3478.
(2) Qian, C.; Li, T.; Shen, T. Y.; Libertine-Garahan, L.; Eckman, J .;
Biftu, T.; Ip, S. Epibatidine is a nicotinic analgesic. Eur. J .
Pharmacol. 1993, 250, R13-R14.
(3) Rupniak, N. M. J .; Patel, S.; Marwood, R.; Webb, J .; Traynor, J .
R.; Elliott, J .; Freedman, S. B.; Flechter, S. R.; Hill, R. G.
Antinociceptive and toxic effect of (+)epibatidine oxalate at-
tributable to nicotinic agonist activity. Br. J . Pharmacol. 1994,
113, 1487-1493.
(4) Sullivan, J . P.; Decker, M. W.; Brioni, J . D.; Donnelly-Roberts,
D.; Anderson, D. J .; Bannon, A. W.; Kang, C. H.; Adams, P.;
Piattoni-Kaplan, M.; Bukley, M. J .; Gopalakrishnan, M.; Wil-
liams, M.; Arneric, S. P. (()-Epibatidine elicits a diversity of in
vitro and in vivo effects mediated by nicotinic acetylcholine
receptors. J . Pharmacol. Exp. Ther. 1994, 271, 624-631.
(5) Cignarella, G.; Barlocco, D.; Tranquillini, M. E.; Volterra, A.;
Brunello, N.; Racagni, G. Interaction of 3,8-diazabyclo[3.2.1]-
octanes with µ and δ opiod receptors. Pharmacol. Res. Commun.
1988, 20, 383-393.
In Vit r o E xp er im en t s. 1. Mem br a n e P r ep a r a t ion s:
Rat cerebral cortical membranes were purchased from ABS
Inc. (Wilmington, DE). Prior to use, the frozen membrane
pellets were slowly thawed, washed, and resuspended in 30
volumes of assay buffer (composition, mM: Tris HCl, 50; NaCl,
120; KCl, 5; MgCl2, 1°, and CaCl2, 2.5; pH 7.4 at 4 °C). The
homogenate was centrifuged at 45000g for 20 min at 4 °C and
the pellet resuspended in ice-cold buffer.
2. [3H]-(-)-Cytisin e Bin d in g: Binding conditions were as
previously described.20 Samples containing 150-200 µg of
protein, 0.7 nM [3H]-(-)-cytisine (30 Ci/mmol), and the various
concentrations of the nAChR modulators were incubated in a
final volume of 500 µL for 75 min at 4 °C in triplicate.
Nonspecific binding was determined in the presence of 10 µM
(-)-nicotine.
3. Tissu e Cu ltu r e: Cells of the IMR-32 human neuroblas-
toma clonal line (ATCC, Rockville, MD) were maintained in a
log phase of growth according to established procedures.21