NSAID Antinociception Measured in a Chemical and a Thermal Assay in Mice

Abstract

The antinociceptive activity of several nonsteroidal anti-inflammatory drugs (NSAIDs) that were administered either intraperitoneally or intrathecally was assessed in mice by two algesiometric tests. The first was the writhing test, which assessed the abdominal constrictions that were induced by intraperitoneal acetic acid (a chemical test), and the second was the tail flick test, which measured pain responses to heat stimuli. The corresponding effective doses and their relative potencies were compared because these tests use different nociceptive stimuli with different transmission pathways. The intraperitoneal and intrathecal dose-response curves for the antinociception induced by every NSAID that was tested were parallel in the writhing test. In the tail flick test, however, only the intraperitoneal and intrathecal dose-response curves for ketoprofen, piroxicam, naproxen, nimesulide, paracetamol and diclofenac were parallel. The results obtained in this study confirm that NSAIDs possess different abilities to induce inhibition of cyclooxygenase, and they can be indirectly assessed by their different antinociceptive activities, depending on the algesiometric assays that are used. The antinociception of most NSAIDs might involve central mechanisms. The findings demonstrate the increasing importance of the spinal cord in processing and modulating nociceptive input, because intrathecal administration of NSAIDs is always more effective (in terms of potency) than systemic administration; thus, the antinociceptive efficacy of NSAIDs strongly depends on the algesiometric assay that is used and on the type of the nociceptive stimulus to which the test subject is exposed.

Full text

ORIGINAL ARTICLE
NSAID antinociception
measured in a chemical and a
thermal assay in mice
HF Miranda PhD, J Lopez MA, F Sierralta DDS, A Correa MA, G Pinardi MD
Pharmacology Program, ICBM, Faculty of Medicine, Universidad de Chile, Santiago, Chile
Correspondence: Dr HF Miranda, Faculty of Medicine, Universidad de Chile, Box 70.000, Santiago 7, Chile. Telephone +56-2-678-6052,
fax +56-2-737-2783, e-mail hmiranda@machi.med.uchile.cl
Received for publication August 30, 2000. Accepted August 29, 2001
HF Miranda, J Lopez, F Sierralta, A Correa, G Pinardi.
NSAID antinociception measured in a chemical and a
thermal assay in mice.
Pain Res Manage 2001;6(4):190-196.
The antinociceptive activity of several nonsteroidal anti-inflam-
matory drugs (NSAIDs) that were administered either intraperi-
toneally or intrathecally was assessed in mice by two
algesiometric tests. The first was the writhing test, which
assessed the abdominal constrictions that were induced by
intraperitoneal acetic acid (a chemical test), and the second was
the tail flick test, which measured pain responses to heat stimuli.
The corresponding effective doses and their relative potencies
were compared because these tests use different nociceptive
stimuli with different transmission pathways. The intraperitoneal
and intrathecal dose-response curves for the antinociception
induced by every NSAID that was tested were parallel in the
writhing test. In the tail flick test, however, only the intraperi-
toneal and intrathecal dose-response curves for ketoprofen,
piroxicam, naproxen, nimesulide, paracetamol and diclofenac
were parallel. The results obtained in this study confirm that
NSAIDs possess different abilities to induce inhibition of
cyclooxygenase, and they can be indirectly assessed by their dif-
ferent antinociceptive activities, depending on the algesiometric
assays that are used. The antinociception of most NSAIDs might
involve central mechanisms. The findings demonstrate the
increasing importance of the spinal cord in processing and mod-
ulating nociceptive input, because intrathecal administration of
NSAIDs is always more effective (in terms of potency) than sys-
temic administration; thus, the antinociceptive efficacy of
NSAIDs strongly depends on the algesiometric assay that is used
and on the type of the nociceptive stimulus to which the test sub-
ject is exposed.
Key Words: Antinociception; Nonsteroidal anti-inflammatory
drugs; Tail flick test; Writhing test
L’antinociception amenée par les AINS telle
que mesurée dans des tests chimique et ther-
mique chez la souris
RÉSUMÉ : L’activité antinociceptive de plusieurs anti-inflam-
matoires non stéroïdiens (AINS) qui ont été administrés soit par
voie intrapéritonéale, soit par voie intrathécale a été évaluée chez
des souris au moyen de deux tests algésiométrique. Le premier a
été le test de torsion qui visait à évaluer les contractions abdo-
minales induites par l’acide acétique intrapéritonéal (test chi-
mique) et le second était le test du battement de la queue qui
mesurait les réponses douloureuses aux stimuli thermiques (test
thermique). Les doses efficaces correspondantes et leur puis-
sance relative ont été comparées, parce que ces tests utilisent des
stimuli nociceptifs différents dont les voies de transmission sont
également différentes. Les courbes dose-réponse intra-péri-
tonéales et intrathécales pour l’antinociception induite par
chaque AINS testé ont été parallèles dans les tests de torsion.
Dans les tests de battement de la queue, par contre, seules les
courbes dose-réponse intra-péritonéales et intrathécales du kéto-
tifène, du piroxicam, du naproxène, du nimésulide, du paracéta-
mol et du diclofénac ont été parallèles. Les résultats obtenus lors
de cette étude confirment que les AINS induisent, à divers
degrés, l’inhibition de la cyclo-oxygénase et qu’ils peuvent indi-
rectement être évalués par leurs activités antinociceptives dis-
tinctes selon les tests algésiométriques utilisés. L’antinociception
de la plupart des AINS pourrait mettre en jeu des mécanismes
centraux. Les conclusions confirment l’importance croissante de
la moelle épinière dans le processus et la modulation de l’influx
nociceptif, parce que l’administration intrathécale des AINS est
toujours plus efficace (sur le plan de la puissance) que l’adminis-
tration systémique. Ainsi, l’efficacité antinociceptive des AINS
dépend fortement du test algésiométrique utilisé et du type de
stimulus nociceptif auquel le sujet est exposé.
190
Pain Res Manage Vol 6 No 4 Winter 2001

N
onsteroidal anti-inflammatory drugs (NSAIDs) are a
group of agents that possess both analgesic and anti-
inflammatory properties. They are used in the treatment of a
variety of pain and inflammatory disorders. There is sub-
stantial evidence that NSAID-induced analgesia is due to
the inhibition of prostaglandin synthesis in both the periph-
eral and central nervous systems (1). Prostaglandins are
generated through two isoforms of the enzyme cyclooxyge-
nase (COX): the constitutive isoform (COX-1) and the iso-
form that is induced at sites of inflammation (COX-2). Most
NSAIDs are principally inhibitors of COX-1, while others
(eg, meloxicam, nimesulide, celecoxib and rofecoxib) have
pharmacological profiles that are clearly different and are
characterized by a more selective inhibition of COX-2 (2).
Meloxicam, compared with other NSAIDs, is the most
potent inhibitor of prostaglandin biosynthesis in pleural and
peritoneal exudates, but is only a weak inhibitor in the gas-
tric tract and kidney. Unlike other NSAIDs, meloxicam
preferentially inhibits inducible human COX-2 in COS cells
(3,4). The activity of nimesulide against COX-1 and COX-2,
measured in vitro and in vivo, demonstrated that it is a
selective inhibitor of COX-2 (5). Several additional sub-
stances have been reported to be potentially involved in the
modulation of the antinociceptive activity of NSAIDs.
These include substance P, nitric oxide, adenosine triphos-
phate, glutamate, serotonin, neurokinin, noradrenaline, ion
channels and acetylcholine (6-13).
Previous studies have demonstrated that NSAIDs can
induce antinociception in several algesiometric tests; how-
ever, a detailed comparative study of the dose-response
characteristics of several NSAIDs in two different algesio-
metric tests has not been performed. The writhing test (vis-
ceral pain) and the tail flick test (somatic pain) are animal
models of acute pain in which the nociceptive stimulus and
the transmission pathways are different (14). The purpose of
the present study was to examine the antinociceptive effect
of NSAIDs that were administered intraperitoneally and
intrathecally in these chemical and thermal algesiometric
assays, to determine their corresponding effective doses and
to discuss their relative potencies.
ANIMALS AND METHODS
CF-1 mice that weighed 25±3 g were used throughout the
study. The animals were acclimated to the laboratory envi-
ronment for a minimum of 2 h before being used, and the
ethical standard guidelines were followed (14). In particular,
the durations of the experiments were as short as possible,
the number of animals involved was kept to a minimum, and
the animals were killed by the administration of an anes-
thetic overdose immediately after the recording period.
Each animal was used only once and received only one dose
of the drugs that were tested. All drugs were freshly pre-
pared by dissolving them in normal saline or in a slightly
hyperbaric solution of glucose (6%), and doses were calcu-
lated on the basis of the drug salts. All observations during
the assay were made in a randomized and blind manner.
Antinociceptive activity was evaluated as previously
reported (15). Briefly, intraperitoneal administration was
accomplished 30 min before the algesiometric test by inject-
ing the total dose in a constant volume of 10 mL/kg. The
Hylden and Wilcox (16) technique was used for intrathecal
administration 15 min before the algesiometric test, and the
doses of the drugs (dissolved in a slightly hyperbaric solu-
tion of glucose [6%] to limit diffusion) were injected in a
constant volume of 5 µL/g. The procedure was performed
rapidly, with a high degree of accuracy and reproducibility.
These times (30 min for intraperitoneal administration and
15 min for intrathecal administration) were found in previ-
ous experiments to be near the time of the onset of maxi-
mum analgesic effect.
Algesiometric tests
Writhing test: Mice were injected intraperitoneally with
10 mL/kg of 0.6% acetic acid, and starting 5 min after the
administration of the acetic acid solution, the number of
writhes during a 5 min period was counted. A writhe was
defined as a contraction of the abdominal muscles, accom-
panied by an elongation of the body and an extension of the
hind limbs. Dose-response curves, which were determined
near the time of peak effect, were constructed to assess the
antinociceptive actions of the different NSAIDs that were
administered either intraperitoneally or intrathecally. Eight
animals were injected at each of four doses to determine a
dose-response curve. The dose that produced 50% of
antinociception (AD
50
: 50% reduction of control writhes)
was calculated by a standard linear regression analysis of
the dose-response curve. Antinociceptive activity was
expressed as a percentage of inhibition of the usual number
of writhes that were observed in control animals that
received intraperitoneal saline (24.3±1.0, n=42) or intrathe-
cal glucose (23.9±0.5, n=42).
Tail flick test: A radiant heat tail flick test algesiometer unit
was used to measure response latencies according to the
method that was described by D’Amour and Smith (17).
Animals responded to a focused heat stimulus by flicking or
removing their inflicted tails from the heat source. The
intensity of the light beam was adjusted so that the control
latency (baseline) readings were approximately 2.5 s, and an
8 s cutoff time was used to prevent tissue damage. Anti-
nociception activity was expressed according to the follow-
ing formula:
% antinociception = 100 × [(test latency-control
latency)/(cutoff time-control latency)]
A dose-response curve was determined by using at least
four doses of each NSAID, and the AD
50
was determined by
linear regression analysis.
All of the drugs used in the study were dissolved in saline
solution when they were administered intraperitoneally or,
in the case of intrathecal administration, in a slightly hyper-
baric solution of glucose (6%) to limit diffusion (with the
exception of ibuprofen and nimesulide, which were dis-
solved in 5 mm dimethylsulphoxide; this vehicle was
NSAID antinociception
Pain Res Manage Vol 6 No 4 Winter 2001
191

Miranda et al
Pain Res Manage Vol 6 No 4 Winter 2001
192
Figure 1) Dose-response curves of the antinociceptive activity of naproxen, clonixin, ibuprofen and diclofenac administered intraperitoneally (i.p.) and
intrathecally (i.t.) in the writhing test in mice
Figure 2) Dose-response curves of the antinociceptive activity of meloxicam, piroxicam, paracetamol and nimesulide administered intraperitoneally (i.p.)
and intrathecally (i.t.) in the writhing test in mice

devoid of antinociceptive activity when tested by itself).
The following drugs, provided by local laboratories in
Chile, were used: ketoprofen (Rhône Poulenc Rorer), pirox-
icam (Pfizer), diclofenac (Novartis), naproxen (Saval),
ibuprofen and clonixine (Pharma Investi of Chile), metami-
zol (dipyrone, Sanderson), paracetamol (Pasteur), meloxi-
cam (Recalcine) and nimesulide (Schering Plough).
Results were presented as AD
50
values with 95% confi-
dence limits (95% CL). Parallelism of the lines that were
obtained by linear regression analysis of the dose-response
curves and relative potencies was calculated according to
the method described by Tallarida and Murray (18), and sig-
nificance was accepted at the 0.05 level.
RESULTS
Effects of NSAIDs in the writhing test
The intraperitoneal or intrathecal administration of the
NSAIDs produced dose-dependent antinociceptive activi-
ties in this chemical algesiometric test. The dose-response
curves are shown in Figures 1, 2 and 3. The intraperitoneal
and intrathecal dose-response curves for each NSAID were
statistically parallel. The corresponding AD
50
(with 95%
CLs) and the relative intraperitoneal/intrathecal potencies
are shown in Table 1. These values vary widely, depending
on the route of administration and the algesiometric test that
was performed. In the writhing test, AD
50
values ranged
from 0.85 to 221 mg/kg after intraperitoneal administration,
and ranged from 0.05 to 9.3 mg/kg after intrathecal admin-
istration.
The order of potency, from greatest to least, of the
antinociceptive activities that were displayed by NSAIDs
(given by intraperitoneal injection) was ibuprofen, meloxi-
cam, diclofenac, nimesulide, piroxicam, clonixine, metami-
zol, ketoprofen, naproxen and paracetamol. The order of
potency, from greatest to least, given by intrathecal injection
was ibuprofen, nimesulide, meloxicam, diclofenac, piroxi-
cam, metamizol, ketoprofen, clonixine, naproxen and para-
cetamol.
Effects of NSAIDs in the tail flick test
In the thermal algesiometric tail flick test, the NSAIDs that
were administered either intraperitoneally or intrathecally
produced dose-dependent antinociceptive activities. The
corresponding AD
50
values and their 95% CLs are shown in
Table 2. Dose-response curves for intraperitoneal and
intrathecal ketoprofen, piroxicam, naproxen, nimesulide,
paracetamol and diclofenac were statistically parallel and
are shown in Figures 4 and 5. The relative potencies (calcu-
NSAID antinociception
Pain Res Manage Vol 6 No 4 Winter 2001
193
Figure 3) Dose-response curves of the antinociceptive activity of metamizol and ketoprofen administered intraperitoneally (i.p.) and intrathecally (i.t.) in
the writhing test in mice
TABLE 1
Dose that produced 50% antinociception (AD
50
) values
with 95% confidence limits (CL) and relative potency of
nonsteroidal anti-inflammatory drugs (NSAIDs)
administered intraperitoneally (IP) and intrathecally (IT) in
the writhing test
AD
50
mg/kg Relative
(95% CL ) Potency
NSAID IP IT IT/IP
Naproxen 46.5 6.5 5.04
(20.7-104.1) (2.1-21.1)
Clonixine 11.3 1.1 9.46
(6.1-21.2) (0.38-3.1)
Ibuprofen 0.85 0.05 16.14
(0.12-6.1 ) (0.02-0.13)
Diclofenac 7.2 0.44 18.08
(2.6-19.9) (0.08-2.6)
Meloxicam 6.5 0.30 20.80
(3.5-11.8) (0.07-1.3)
Piroxicam 9.0 0.51 20.85
(3.3-24.1) (0.38-0.69)
Paracetamol 221.0 9.3 21.33
(140-346) (1.9-10.8)
Nimesulide 7.6 0.27 24.25
(3.7-15.4) (0.21-0.39)
Metamizol 28.5 0.79 24.92
(19.8-41.1) (0.4-1.5)
Ketoprofen 30.3 0.86 30.33
(11.3-81.6) (0.28-2.6)
The drugs are listed according to the increment in the relative potency

lated by the intrathecal/intraperitoneal ratio) of the NSAIDs
in which dose-response curves were parallel are summa-
rized in Table 2.
The order of potency, from greatest to least, of the
antinociceptive activities displayed by NSAIDs that were
given by intraperitoneal injection was nimesulide, ketopro-
fen, ibuprofen, meloxicam, clonixine, piroxicam, naproxen,
diclofenac, metamizol and paracetamol. Given by intrathe-
cal injection, the order of potency, from greatest to least,
was meloxicam, ketoprofen, diclofenac, nimesulide, clonix-
ine, naproxen, piroxicam, paracetamol, metamizol and
ibuprofen.
DISCUSSION
The results that were obtained in this study confirm that
NSAIDs possess different capacities to induce the inhibition
of COX, as reflected in their different antinociceptive activ-
ities in two nociceptive tests. In the tail flick test, intraperi-
toneal administration resulted in an AD
50
range of 28.6 to
572.2 mg/kg and intrathecal administration resulted in an
AD
50
range of 1.18 to 221.9 mg/kg. The dose-response
curves for intraperitoneal and intrathecal ibuprofen, clonix-
ine, meloxicam and metamizol, however, significantly devi-
ated from parallelism, which suggested that the
antinociceptive activities of these NSAIDs were produced
by the activation of different peripheral and spinal pathways
(19). In the chemical nociceptive test, ibuprofen was the
most potent drug administered either intraperitoneally or
intrathecally, while it was clearly less potent in the tail flick
test. Ibuprofen has been characterized as a purely competi-
tive inhibitor of COX-1 and COX-2, producing a readily
reversible effect (20). On the other hand, naproxen, which
was a low potency analgesic in the writhing test, is a more
selective COX-2 inhibitor. Naproxen shows a time-depend-
ent effect (involving a second step), which occurs within
seconds to minutes and may reflect the induction of a pro-
tein conformational change with the formation of a complex
in which the inhibitor is bound more tightly to the enzyme
(20). Because the tail flick test did not involve an inflamma-
tory component, COX-2 induction should not have been
present, and time-dependent inhibitors should conceivably
have exhibited lower antinociceptive activities.
In the rat spinal cord/tail in vitro preparation, very low
doses of paracetamol (10 to 50 µM) induced inhibition of
COX and blocked bradykinin-activation of nociceptors
(21). Even if it is not possible to establish a direct pharma-
cological correlation with this report (because of the nature
of the assays), paracetamol-induced antinociception with
algesiometric tests in the present study was evident at doses
that were four to 76 times higher than in other studies (6),
which strongly suggests the involvement of central neural
mechanisms in addition to COX inhibition.
In a study of NSAID suppression of evoked nociceptive
activity in individual thalamic neurons, Jurna (22) obtained
low AD
50
values for diclofenac and ibuprofen; these results
support the idea that central effects of NSAIDs contribute to
their analgesic efficacy because they do not completely
inhibit prostaglandin biosynthesis. The findings on the
effects of diclofenac and ibuprofen in the present acetic acid
writhing test are comparable with these previous results and
with those reported by Bjorkman (6), who demonstrated that
diclofenac induced antinociception in visceral pain models
(writhing test) but not in somatosensory models (tail flick
test).
Reports that ketoprofen is more potent than diclofenac as
an analgesic agent in vivo (23) do not agree with the results
of the writhing test in the present study. This finding could
be explained by differences in experimental models – the
route of administration (oral and intravenous compared with
intraperitoneal and intrathecal) and the active enantiomers
that were used (dexketoprofen compared with racemic keto-
profen).
The two models of animal pain that were used in this
study have been used extensively over time. They represent
different models of acute pain because the writhing test is a
chemical assay that may produce some inflammation and is
used as a model of visceral pain, while the tail flick test is a
somatic pain model, without inflammation. NSAIDs are less
effective (24,25) in the tail flick test than in the writhing
test. Our results show that, irrespective of the mechanisms
related to the quality and intensity of the pain stimuli, the
NSAIDs always elicited antinociception in both tests.
Miranda et al
Pain Res Manage Vol 6 No 4 Winter 2001
194
TABLE 2
Dose that produced 50% antinociception (AD
50
) values
with 95% confidence limits (CL) and relative potency of
nonsteroidal anti-inflammatory drugs (NSAIDs)
administered intraperitoneally (IP) and intrathecally (IT) in
the tail flick test
AD
50
mg/kg Relative
(95% CL ) Potency
NSAID IP IT IT/IP
Ketoprofen 43.7 4.3 8.16
(12.6-151.6) (1.6-11.6)
Piroxicam 160.2 25.5 13.50
(64.4-398.2) (1.3-509.4)
Naproxen 218.1 10.0 17.06
(58.9-807.7) (2.7-37.8)
Nimesulide 28.6 5.0 18.91
(9.4-86.8) (3.0-8.3)
Paracetamol 572.2 28.4 32.64
(172-1903) (13-61.8)
Diclofenac 424.1 4.5 33.61
(60.5-2972) (1.4-14.3)
Ibuprofen 57.3 221.9 N/A
(21.9-149.6) (197.5-296.3)
Clonixine 118.0 8.9 N/A
(62.8-221.6) (1.2-64.5)
Meloxicam 100.4 1.2 N/A
(42.3-238.5) (0.6-2.4)
Metamizol 467.4 179.8 N/A
(423.1-516.3) (35.9-900.4)
The drugs are listed according to the increment in the relative potency.
NA Not applicable

NSAID antinociception
Pain Res Manage Vol 6 No 4 Winter 2001
195
The results of this study indicate that antinociception
after the administration of most NSAIDs might involve cen-
tral neural mechanisms, which add to the peripheral
antinociception that is induced by COX inhibition. These
findings demonstrate the increasing importance of the spinal
cord in processing and modulating nociceptive input,
because intrathecal injections of NSAIDs are always more
effective in terms of potency than systemic administration.
The antinociceptive efficacy of NSAIDs, therefore, strongly
depends on the algesiometric assay that is used and on the
type of the nociceptive stimulus to which the test subject is
exposed.
ACKNOWLEDGEMENTS: This work was supported by grant
number 1990842 from FONDECYT, Chile.
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M ED IATO RS
INFLAMMATION
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Behavioural
Neurology
Endocrinology
International Journal of
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Disease Markers
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BioMed
Research International
Oncology
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Oxidative Medicine and
Cellular Longevity
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PPAR Research
The Scientic
World Journal
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Immunology Research
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Journal of
Obesity
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Computational and
Mathematical Methods
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Ophthalmology
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Diabetes Research
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Research and Treatment
AIDS
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Gastroenterology
Research and Practice
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Parkinson’s
Disease
Evidence-Based
Complementary and
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