Anti-inflammatory effect of a new piperazine derivative: (4-methylpiperazin-1-yl)(1-phenyl-…
Ò
(
Sigma Chemical, USA), dexamethasone (Decadron ,
mirror was placed under this box to enable the unhindered
observation of the formalin-injected paw for 30 min. Pain
reaction time (licking time) was assessed during two
periods: from 0 to 5 min, the first phase, where neurogenic
pain is caused by direct stimulation of the nociceptors, and
from 15 to 30 min, the second phase, where inflammatory
pain is caused by the release of inflammatory mediators.
These results were expressed as the mean ± SEM of
licking time in seconds.
Ache, Brazil), formaldehyde (Synth, Brazil), indomethacin
Ò
(
Indocid , Merck Sharp & Dohme Farmac eˆ utica-Ltda, SP,
Ò
Brazil), and morphine hydrochloride (Dimorf , Cristalia,
SP, Brazil). LQFM182 (2) was dissolved in 10% DMSO in
distilled water, and all other drugs were dissolved in dis-
tilled water. The doses of the LQFM182 (2) were based on
the doses of JNJ7777120 (1) used in previous studies
(
Hsieh et al. 2010).
Anti-nociceptive activity
Anti-inflammatory activity
Acetic acid-induced abdominal writhing test The acetic
acid-induced nociception was achieved as described pre-
viously by Koster et al. (1959). Groups of mice (n = 8)
were treated by gavage (p.o.) with vehicle (10% DMSO
Carrageenan-induced oedema Carrageenan-induced hind
paw oedema in mice was used as the animal model of acute
inflammation according to the method of Winter et al.
(1962). Initially, the animals (n = 8) were treated with
vehicle (10% DMSO, 10 mL/kg, p.o.), LQFM182 (2)
(100 mg/kg, p.o.) or indomethacin (10 mg/kg p.o.—posi-
tive control). One hour later, 50 lL of carrageenan (1%)
was injected intraplantar into the right hind paw and 50 lL
of saline (0.9% NaCl) into the left hind paw (used as the
control). The paw volume was measured using a plethys-
mometer (Model 7141, Ugo Basile, Italy) at 1, 2, 3 and 4 h
after the injection of carrageenan. The baseline was per-
formed before the treatments (time 0) for each animal. The
results were expressed in lL as mean ± SEM.
1
0 mL/kg), LQFM182 (2) at doses 50, 100 or 200 mg/kg
or indomethacin (10 mg/kg, positive control for anti-no-
ciceptive activity) 60 min before the application of acetic
acid solution (1.2% v/v; 10 mL/kg, i.p.). The number of
abdominal constrictions (writhing) was counted for each
animal, over a period of 30 min after acetic acid injection;
the results are expressed as the mean ± SEM of the
number of writhings.
Tail flick test The tail flick test was performed as
described previously by D‘Amour et al. (1941). In this test,
the time taken to flick the tail (latency) when it was
exposed to a heat source was measured using an analges-
Carrageenan-induced pleurisy The animals (n = 8) were
treated with vehicle (10% DMSO, 10 mL/kg, p.o.),
LQFM182 (2) (100 mg/kg, p.o.) or dexamethasone (2 mg/
kg, p.o.—positive control). One hour after the treatments,
the animals received an injection of 100 lL of 1% car-
rageenan into the pleural cavity. Four hours later, the
pleural exudate was collected with 1 mL of heparinised
phosphate-buffered saline and used to count the number of
total leukocytes using T u¨ rk solution in a Neubauer cham-
ber (Vinegar et al. 1973), to evaluate the myeloperoxidase
activity, the levels of TNF-a and IL-1b.
Ò
imeter (Insight , Ribeir a˜ o Preto, S a˜ o Paulo, Brazil). The
animals were divided into three experimental groups
(
(
n = 8): vehicle (10% DMSO, 10 mL/kg, p.o.), LQFM182
2) (100 mg/kg, p.o.) or morphine (5 mg/kg, s.c.—positive
control for anti-nociceptive activity). The latency baseline
was performed 30 min before the treatments (time -30)
and immediately after of the treatments (time 0) for each
animal. After, the latency to pain reaction was measured at
3
0, 60, 90, 120 and 150 min. A cut-off of 15 s was set to
prevent the risk of burns. The results were expressed as
mean ± SEM, in seconds, at the different times.
Activity of myeloperoxidase enzyme To measure the
myeloperoxidase (MPO) activity, 40 lL of pleural lavage
of mice treated with vehicle (10% DMSO, 10 mL/kg, p.o.),
LQFM182 (2) (100 mg/kg, p.o.) or dexamethasone (2 mg/
kg, p.o.—positive control) was transferred to eppendorfs.
The reaction was started by adding 360 lL of phosphate
buffer pH 6.0 containing 0.167 mg/mL of o-dianisidine 2
HCl and 0.0005% H O . The enzyme reaction was stopped
Formalin test Formalin-induced nociception was per-
formed as described previously by Hunskaar and Hole
(
1987). Groups of mice (n = 7) were treated with vehicle
10% DMSO 10 mL/kg p.o.), LQFM182 (2) (100 mg/kg
(
p.o.), indomethacin (10 mg/kg, p.o.—positive control for
anti-nociceptive activity in the second phase), or morphine
2
2
(
5 mg/kg, s.c.—positive control for anti-nociceptive
after 15 min by adding 30 lL of sodium azide 1%. The
samples were centrifuged subsequently for 5 min at
1000 rpm. The supernatant was separated and 200 lL was
transferred to microplate wells; absorbance was assayed by
enzyme-linked immunosorbent assay (ELISA), at a wave-
length of 450 nm (Sedgwick 1995; Saleh et al. 1999). The
activity in the first and second phases). Sixty min after the
p.o. treatment, or 30 min after s.c. treatment, 20 lL of 3%
formalin (in saline) was administered into the plantar sur-
face of the right hind paw. After the phlogistic agent
injection, the mice were placed into an acrylic box, and a
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