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Introduction
Humans have been using non-steroidal
anti-inflammatory drugs (NSAIDs) for more than 3500 years. The first real
progress in our understanding of the mechanism of the
action of NSAIDs occurred 30 years ago, when it was revealed
that these chemically varied drugs reduced the formation of
prostaglandins. This ability is associated with the inhibition
of cyclooxygenase (COX), which converts arachidonic acid
to its precursor prostaglandin (PG) H2. Two isoforms of COX
have been identified. The constitutively expressed COX-1
is the housekeeping enzyme mainly responsible for the
physiological activities of prostaglandins, whereas COX-2, whose
expression is mainly induced under inflammatory conditions,
is responsible for pathological prostaglandins that produce
pain and fever[1]. On the basis of these observations,
COX-2-selective drugs such as celecoxib were rapidly developed
and became one of the most commercially successful classes
of drugs[2].
We synthesized 13 pyrazole derivatives based on the
chemical structure of celecoxib[3,4], including PC-406 and
PC-407, which are derivatives of celecoxib created by changing
the group at the 5 position in the pyrazole ring. For PC-406,
the phenyl structure at the 5 position is replaced by an
isopropyl group; for PC-407, it is replaced by a naphthyl group
(Figure 1). The chemical structures of these derivatives have
been confirmed by infrared absorption spectrum, ultraviolet absorption spectrum, 1H nuclear magnetic resonance,
13C nuclear magnetic resonance and mass spectrum analysis.
The purity values of PC-406 and PC-407 were found to be
99.5% and 97.5%, respectively, by high performance liquid
chromatography. Using the mouse ear edema model, PC-406
and PC-407 were found to have more potential activity than
celecoxib in anti-inflammation. PC-406 and PC-407 were also
observed to have fewer gastrointestinal side effects. In the
present study, we tried to evaluate the analgesic effects and
inhibitory effects on COX-1/COX-2 of candidate compounds.
Materials and methods
Animals BALB/c mice obtained from the Experimental
Animal Center of the Fourth Military Medical University were
used. Half of the mice were males and half were females. All
the animals weighed 18-22 g. Animals were housed in
colonial stock following arrival. Food and water were available
ad libitum. Temperature and humidity of the environment
were controlled (23±1°C and 50.0%±1.3%, respectively), and
a 12 h day/night cycle was used. All the experiments were
carried out during the light phase. The experimental design
was approved by the relevant ethics board of the institution
in which the study was carried out.
Drugs and reagents PC-406 and PC-407 were
synthesized by our group. The celecoxib was a gift from Pharmacia
(Rockville, MD, USA). Lipopolysaccharide (LPS) and
calcimycin were purchased from Sigma (St Louis, USA). The
Brewer thioglycollate medium was from (Difco
Company, Detroit, USA). Newborn calf serum and RPMI-1640 were
from (Hyclone, Logan, USA). The
6-keto-PGF1a and PGE2 radioimmunoassay (RIA) kits were from Chemclin Biotech
(Beijing, China). For the analgesic effect assays, all the test
compounds were dissolved in 3% Tween-80 and
administered intragastrically in a volume of 0.3 mL. For the
selectivity evaluation, all the tested samples were prepared in stock
solution (0.01 mol/L) with Me2SO and stored at -20 °C.
Before use, the stock solution was diluted to a series of
concentrations in RPMI-1640.
Antinociceptive assays Four antinociceptive assays were
used: the writhing test, hot tail-flick test, hot plate test, and
formalin test. Mice were fasted for 24 h with water given
ad libitum, maintained at room temperature and were divided
into 4 groups (n=6): control group, CEL group, PC-406 group,
and PC-407 group, which received pretreatment with 3%
Tween-80, celecoxib, PC-406, and PC-407, respectively. The
2nd, 3rd, and 4th groups were further divided into 3
subgroups (A,B,C), which received test compounds at 100, 50,
and 25 mg/kg, respectively. Celecoxib was used as a
positive control.
Writhing test All the pretreatments were carried out 1 h
prior to intraperitoneal injection of 0.1 mL/10 g of 0.6% acetic
acid, which caused a typical writhing response. The number
of writhing responses was counted for 10 min by observers
who were blinded to the treatment. The antinociceptive
effects of drugs were measured by calculating the mean
reduction in the number of abdominal constrictions for each drug,
as compared to that produced by the Tween-80 vehicle.
Hot tail-flick test The antinociceptive effects of the test
substances were determined by the hot tail-flick method as
described by Sewell and Spencer[5]. The tails of mice (1-2
cm) were immersed in warm water kept constant at 53 °C, and the
reaction time was measured as the time taken for the mice to
deflect their tails. The first reading was ignored, and the
reaction time was taken as the mean of the next 2 readings.
The latent period until the tail-flick response was taken as
the index of antinociception and was determined before and
1 h after the administration of drugs. The maximum reaction
time was fixed at 3 s. The analgesia percentage (AP) was
calculated as: AP=(ttest
reaction-tcontrol
reaction)/(3-tcontrol
reaction).
Hot plate test The method used has been described by
Baker et al[6]. A metal hot plate was heated to a constant
temperature. Behavioral measurements were taken at 55±0.5 °C.
The temperature of the plate was monitored at all times. To
confine the animals to a certain observation area, a colorless
acrylic cylinder of 20 cm diameter was placed on the hot
plate. After each measurement the plate was wiped with a
damp cloth to remove traces of urine and feces. Latency for
the animal to lick its hindpaw was measured before and 1 h
after pretreatment. The offset time was set at 30 s. AP was
calculated as: AP=(ttest
reaction-tcontrol
reaction)/(30-tcontrol
reaction).
Formalin test Formalin solution (5 %, 50 mL) was injected
subplantarly into the left hindpaws of the mice using a
microsyringe with a 26-gauge needle. Animals were then
placed in a Plexiglas chamber and observed for nociceptive
behavior for 1 h. The amount and duration of licking or
flinching were recorded at 5 min intervals over the next 60
min. The sum of the amount and duration of licking or
flinching in the early phase (0-10 min, phase 1) and late phase
(20-60 min, phase 2) were calculated, respectively. To calculate
the ED50, the decrease in the summed amount of licking or
flinching during the whole observation period as compared
with the control group was calculated.
Cell culture Adherent macrophages were harvested from
the peritoneal cells of male mice 3 d after the intraperitoneal
injection of Brewer thioglycollate medium (50 mL/kg).
Peritoneal cells obtained from 2-3 mice were mixed and cultured
in RPMI-1640 supplemented with 5% new-born calf serum.
After settlement for 2 h, non-adherent cells were washed
with cold phosphate-buffered saline (PBS). Then,
macrophages were seeded in 96-well cell culture clusters (Gibco, USA)
at a cell density of 1×109/L. Almost all the adherent cells
were macrophages as assessed by Giemsa staining. Cell
viability was examined by Trypan blue dye exclusion. All
the incubation procedures were performed with 5%
CO2 in humidified air at 37 °C.
COX-1 assay Macrophages were incubated with test
compounds at different concentrations or with the solvent
(Me2SO) for 1 h and were stimulated with calcimycin (1
mmol/L) for 1 h. The same volume of RPMI-1640 was added for the
control groups. The amount of
6-keto-PGF1a in the supernatant was measured by RIA, using the manufacturer¡¯s
instructions. The inhibitory ratio (IR) was calculated as IR =
(Ccalcimycin-Ctest
compound)/(Ccalcimycin
-Ccontrol), where C is the
concentration of 6-keto-PGF1a in the supernatants of the
calcimycin, test compound or control groups.
COX-2 assay Macrophages were incubated with test
compounds at different concentrations or with the solvent
(Me2SO) for 1 h and were stimulated with LPS (1 mg/L) for
9 h. The same volume of RPMI-1640 was added for the control groups. The amount of PGE2 in the supernatant was
measured by RIA, using the manufacturer¡¯s instructions.
The IR was calculated as IR=(CLPS
-Ctest
compound)/(CLPS-C
control), where C is the concentration of
PGE2 in the supernatants of the LPS, test compound or control groups.
Statistics Data are presented as mean±SD of
n observa-tions. The 95% confidence limits of the
ED50 and IC50 values were also calculated. Comparisons between the groups were
carried out by using one-way analysis of variance (ANOVA),
followed by the least significant difference multiple
comparisons test. A P-value of 0.05 was considered statistically
significant. Dose-inhibitory effect curves were fitted by
using sigmoidal dose-response curves (variable
slope)(inhibitory effect on COX) or linear regression (analgesic
effect) using Graphpad Prism (version 4.00, GraphPad
Software Inc , San Diego, USA).
Results
Acetic acid-induced writhing Intraperitoneally injected
acetic acid produced abdominal constrictions, which were
characterized by a stretching response. The mean number
of writhing motions observed in the animals of the control
group over 10 min was 10.33±6.03. All the 3 test compounds
at a high dose (100 mg/kg) significantly (P<0.05) reduced the
number of writhing motions to 5.3±4.0, 1.3±2.3, and 1.7±1.5,
respectively. However, test compounds at low doses (50 or
25 mg/kg) did not significantly reduce the number of
writhing motions induced by acetic acid (Figure 2). The
ED50 values of celecoxib, PC-406 and PC-407 for acetic
acid-induced writhing were 94.2, 67.9, and 63.3
mg/kg respectively (Figure 3, Table
1).
Hot tail-flick response The animals in the control group
did not show any significant effects with respect to the
latent period until tail-flick response. The antinociceptive
effect of PC-406 at a high dose (100 mg/kg) was evident
(P<0.05) and the duration of the tail-flick response increased
from 0.87±0.27 to 1.92±0.15 s. PC-406 at lower doses (25 or 50
mg/kg) did not have any significant analgesic effect as
measured by this assay. However, PC-407 at all doses produced
significant antinociception (P<0.05, Figure 5). The
ED50 values of celecoxib, PC-406, and PC-407 as assessed by the hot
tail-flick assay were 104.7, 89.1, and 30.0 mg/kg, respectively
(Figure 4, Table 1).
Hot plate response Animals in the control group did not
show any significant effects with respect to the latent period
until hot plate response. Both PC-406 and PC-407 produced
a significant analgesic effect as measured by the hot plate
response (P<0.05, Figure 5). The
ED50 values of celecoxib,
PC-406 and PC-407 as measured by hot plate response were
50.6, 46.1, and 76.8 mg/kg, respectively (Figure 6, Table 1).
Formalin test All the mice exhibited elevating, shaking,
licking or biting behaviors in the injected left hindpaw after
subcutaneous injection of formalin into the plantar surface.
These behaviors had 2 distinct phases: an early phase (phase
1), which appeared immediately after formalin injection and
lasted 10 min, and a late phase (phase 2), which appeared
about 20 min after injection and lasted 40 min. PC-406 and
PC-407 (25-100 mg/kg) elicited a dose-dependent inhibitory
effects on licking time, with a maximal reduction of
approximately 85% of the control response
(n=8). For animals treated with PC-406 or PC-407, the durations and amounts of
flinching or licking were significantly decreased
(P<0.05, Figures 8, 9). The
ED50 values of celecoxib, PC-406 and PC-407 were
67.1, 55.8, and 68.8 mg/kg, respectively (Figure 10, Table 1).
COX inhibitory effect The
IC50 values of celecoxib and PC-407 for COX-1 were 39.8 nmol/L and 27.5 nmol/L,
respectively. For PC-406, the IC50 value was more than
10 000 nmol/L (Figure 11A). The
IC50 of test compounds for COX-2 were 4.78, 8.9, and 1.9 nmol/L, respectively (Figure
11B). The selectivity of the test compounds for
COX-1/COX-2 was expressed as the value of
IC50,COX-1/IC50,COX-2. PC-406 showed a 112.2-fold preference for COX-2 versus
COX-1 (Table 2).
Discussion
The discovery and characterization of the COX-2 enzyme
early in the 1990s led to many studies on selective inhibitors
of this isoform. It was hypothesized that COX-2 selective
inhibitors would exhibit similar clinical efficacy but reduced
ulcerogenicity relative to traditional NSAIDs. Less than a
decade after the discovery of COX-2, celecoxib and rofecoxib
were launched, and were very effective analgesics and
anti-inflammatory agents. Furthermore, in recent years, coxibs
have been proved to have potential in the treatment of
cancer and Alzheimer¡¯s disease. All things considered, the
prospects for COX-2 selective inhibitors are good. Based on the
chemical structure of celecoxib, we synthesized PC-406 and
PC-407, 2 pyrazole derivatives, by retaining
benzenesulfona-mide, the necessary active component of the molecule. In
the present study, their analgesic effects and selectivity for
COX-2 were evaluated.
All the antinociceptive assays showed that derivatives
of celecoxib made by substituting groups at the 5 position
exhibited analgesic effects. However, the results from
different assays were different. Four antinociceptive assays were
used in our study. Among them, the hot plate and hot tailflick tests test responses to thermal stimulations, whereas
the writhing and formalin tests test responses to chemical
stimulations. The pain pathways these tests focus on vary
as well. The formalin and hotplate tests mainly focus on
changes above the spinal cord level, whereas the tail flick
and writhing tests emphasize changes at or under the spinal
cord level. In the present study, the results of both the hot
plate test and the formalin test show that PC-406 has the
lowest ED50 value. However, the results of the writhing and
tail flick test show that PC-407 has the lowest
ED50 value. Thus we can infer that PC-406 exerts more effect on the
pathways above the spinal cord level, whereas PC-407 exerts
more effect on pathways peripherally.
Detailed data from the formalin test showed the same
results. The formalin test is a well characterized and
validated evaluation method in mice and rats. It is an accepted
method for the rapid and easy screening of pharmacological
targets in drug evaluation, because it can be performed in
small animals, and the time course of the noxious stimulation
is limited to 1 h. Subplantar injection of formalin results in
flinching, licking or biting behavior during an early acute
phase, which resembles acute pain, followed by a quiescent
interphase, and then a second delayed phase representing
chronic pain. The acute phase is believed to correspond to
the peripheral pain pathways, whereas the second phase
represents more central pain processing mechanisms. In our
study, both phases of the formalin test were well delineated
and could be quantified. Celecoxib and its 2 derivatives
could significantly decrease both the duration and amount
of pain behaviors during the formalin test
(P<0.05). Celecoxib and PC-407 had more effect on behaviors in phase 1, whereas
PC-406 affected behaviors in phase 2 more. That is, celecoxib
and PC-407 affected the peripheral pain pathways more,
whereas PC-406 affected the central pain pathways more.
This is consistent with what we found in our analysis of
different ED50 values.
The differences in the different analgesic effects of the
test compounds are related to their different structures. So
we subsequently investigated the relationships between the
modifications of their chemical structures and their
selectivity for COX-2. To evaluate the selectivity of compounds for
COX-2, many models have been
established[7-10]. Macrophages are known to release prostanoids in 2 kinetically
distinct patterns: the immediate and delayed
phases[11]. In the immediate phase,
6-keto-PGF1a and TXB2 are the major
arachidonic acid metabolites. However, in the delayed phase,
PGE2 is produced depending on induced COX-2. Macrophages
can spontaneously secrete low levels of
6-keto-PGF1a and PGE2. Stimulation with calcimycin or LPS promotes a dra
matic enhancement of prostanoid production. Shen
et al developed a whole-cell assay based on murine peritoneal
macrophages and demonstrated that it was appropriate for
drug design-oriented in vitro
assay[12]. This method was used in the present study.
The results of selectivity evaluation show that PC-407
and PC-406 provide stronger selectivity with respect to the
inhibition of COX-2 than does celecoxib. PC-406 has the
greatest selectivity. It might be the reason for their weak
influence on stomach mucosa. The present study indicates
that test compounds with a non-aryl group or an aryl group
substituted at the 5 position in the pyrazole ring still retain
their anti-inflammatory activity and their ability to inhibit
COX-2 selectively. Furthermore, substitution at the 5
position with a non-aryl group might be more beneficial with
respect to COX-2 selectivity than substitution with an aryl
group.
It has been proved that COX-2 plays a more central role
and is more highly expressed in the central nervous system
than is COX-1, which is the basis of most of its therapeutic
effects. The higher selectivity for COX-2 of PC-406
contributes to its central pain pathway effects. However,
identification of the exact parts of the pain pathways that the 2
derivatives affect needs further investigation.
In conclusion, derivatives of celecoxib made by
substituting with either a isopropyl or a naphthyl group at the 5
position in the pyrazole ring produced analgesic effects with
respect to both thermal and chemical nociception. However,
substitution with an isopropyl group improves the effect on
the central pain pathway better, whereas substitution with a
naphthyl group affects the peripheral pathway more. This is
correlated with the altered selectivity for COX-2. Both
derivatives had greater selectivity than celecoxib. However,
substitution with an isopropyl group was more beneficial
with respect to COX-2 selectivity than substitution with an
naphthyl group.
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