Key words
anti-inflammatory drugs - pharmacology - inflammation
Introduction
Pain is a considerable unpleasant sensory experience that may be severe to moderate,
with transitory or plain intensity and transitory or persistent duration, characteristics
that are associated with different types of pain, whether nociceptive, inflammatory
or neuropathic. There are different animal models to evaluate pain among them, the
formalin test should be mentioned [1]. The formalin test has been widely used in pain investigations, since it was reported
by Dubuisson and Dennis [2]. The assay in which a dilute solution of formalin is injected into the dorsal or
plantar hindpaw of a murine produces two ‘phases’ of pain behavior separated by a
inactive period. The early phase (Phase I) is probably due to direct activation of
nociceptors through TRPA1 (transient receptor potential ankyrin subtype1 protein)
channels and the second phase (Phase II) is due to ongoing inflammatory input and
central sensitization [3].
The formalin test, for antinociception, has been evaluated as an essay that , associates
moderate, continuous pain generated by injured tissue and differs from most traditional
tests of nociception which rely upon brief stimuli of threshold intensity [3]. This test is widely used because some of its events appear to be similar to the
clinical pain of humans. The analysis of the mechanism of action of the formalin test
has been the subject of numerous studies demonstrating the role of most pronociceptive
modulators, such as norepinephrine, serotonin, substance P, neuroquinines, cytokines,
acetylcholine, glutamate, capsaicin, nitric oxide and many others [4].
Among the pronociceptive mediators of the formalin hind paw assay is the nitric oxide
(NO). NO is a molecular gas generated by the activity of nitric oxide synthase (NOS).
Three different forms of NOS have been identified: neuronal NOS (nNOS, type 1 NOS);
inducible NOS, (iNOS, type 2 NOS); endothelial NOS (eNOS, type 3 NOS) and in some
tissues they may exist in the mitochondria (mNOS). nNOS contributes to the NO nociception
by upregulation of the spinal cord after noxious stimulation. Besides, pharmacologic
inhibition of nNOS attenuates nerve injury-induced mechanical hypersensitivity in
mice. iNOS repair of injured tissue and is involved in the development of hypersensitivity
to pain in inflammatory and neuropathic pain models. NO generated by eNOS may modulate
acute tissue inflammation. It has been reported that NO produced in excess by iNOS
and nNOS has been implicated in processes such as pain and inflammation [5]
[6]. It should be remembered that several studies have shown that NO is able to induce
pro- or anti-nociception depending on the locally concentrations of NO produced locally,
the via of administration or application [7]
[8].
On the other hand, the NO produced by the three isoforms of NOS, is subject to inhibition
that is beneficial in shock, inflammation and neurolesion. Pharmacological inhibition
of NOS can be carried out by different agents, including arginase, calmodulin, amino
guanidine, NG-nitro-L-arginine methyl ester (L-NAME), 7-nitro-indazole and others. The antinociception
induced by these agents it has been informed. Moore et al., 1991 and 1993 [9]
[10] reports that the last two nitric oxide biosynthesis inhibitors, produces antinociceptive
activity. This findings provides support that NO is a mediator of pain. It was reported
that L-NAME, induced a dose-dependent stereospecific inhibition of the second phase
of formalin paw test, with minimal effect on the first phase [11]. In addition, pretreatment with L-NAME significantly reduced licking behavior induced
by formalin into the left hindpaw. The results suggest that NO is associated with
nociception. Besides, L-NAME produced a dose-dependent antinociceptive activity in
the acetic acid writhing test, intraplantar paw carrageenan [12]. It has also been reported that NOS inhibitors: L-NAME, 7-nitro-indazole and amino
guanidine reduced nociception of phase II of the formalin hind paw, without modify
phase I, with the exception of L-NAME that decreased it [13].
It is recognized that the nociceptive activity of formalin is directly dependent on
the relative concentration injected in the hind paw.. Low concentrations of formalin
(0.125 and 0.5 %) produce only the neurogenic phase or phase I, acute or phasic. Administration
from 2% of formalin induced also the inflammatory phase or phase II, tonic. Pretreatment
with L-NAME resulted in a significant inhibition of the paw-licking response induced
by both concentrations of formalin [14]. On the other hand, the administration of 7-nitro-indazole induced antinociceptive
activity, in the same animal pain model.
According to the previous backgrounds, L-NAME and 7-nitroindazole, have properties
that allow it to act either as a pronociceptive agent or as an antinociceptive agent,
against different agents. Established this dichotomy, in this work was evaluated whether
the effect of L-NAME and 7-nitroindazole are involved in the antinociceptive activity
of NSAIDs using the formalin test in mice paw.
Materials and Methods
Animals
Male CF-1 mice (28–30 g), housed on a 12 h light–dark cycle at 22±2°C with access
to food and water ad libitum, were used. Experiments were performed in accordance
with current Guidelines for The Care of Laboratory Animals and Ethical Guidelines
for investigation of experimental pain approved by the Animal Care and Use Committee
of the Faculty of Medicine, University of Chile. Animals were acclimatized to the
laboratory for at least 1 h before testing, used only once in the protocol and were
euthanized by intraperitoneal (i.p.) injection of 65 mg/kg of pentobarbital. In each
protocol was used a minimum mice (6–8) to reach definitive results of the drug treatments.
Nociceptive test
Antinociception was assessed by the formalin hind paw assay as previously described
[15], using 20 µL of 2% formalin solution injected into the dorsal surface of the right
hind paw of the mice with a 27-gauge needle attached to a 50-μL Hamilton syringe.
The degree of pain intensity was assessed as the total time, in seconds, spent by
the animal licking or biting the injected paw. The test shows two clear-cut periods:
phase I corresponding to the 5-min period starting immediately after the formalin
injection and described as phasic and neurogenic, resulting from direct activation
of chemical nociceptors, whereas phase II, inflammatory, recorded as the 10-min. period
starting 20° min after the formalin injection and responses result from the central
sensitization of nociceptive pathways leading to motor responses. Control saline animals
licking were 126.38±8.48 and 155.65±10.20 phase I and phase II, respectively and n=24
for each phase). The licking times observed were converted to % of maximum possible
effect (% MPE) as follows:+
% MPE=100− [(100 × post-drug licking time)/control licking time]
Protocol
In order to determine the relative potency antinociceptive de each NSAIDs, dose–response
curves, were obtained for piroxicam (1,3,10,and 30 mg/kg), parecoxib (0.3, 1,3,10
and 30 mg/kg), dexketoprofen (3,10,30 and 100 mg/kg) and ketoprofen (3,10,30 and 100 mg/kg)
were obtained using at least six to eight animals at each of at least four doses administered
i.p. Dose-response for each NSAIDs were created before and after 5 mg/kg of L-NAME
i.p. or 5 mg/kg i.p. of 7-nitroindazole. A least-squares linear regression analysis
of the log dose–response curves allowed the calculation of the dose that produced
50% of antinociception (ED50) for each drug.
Drugs
The drugs were freshly dissolved in a physiological salt solution of 10 mg/kg for
i.p. administration. Piroxicam was provided by Pfizer Chile, parecoxib by Pfizer Chile,
ketoprofen by Rhone- Poulenc Rorer, dexketoprofen was a gift from Menarini Laboratories,
Spain. Nw-nitro-L-arginine methyl ester hydrochloride (L-NAME) and 7-nitroindazol,were purchased
from Sigma-Aldrich Chemical Co, St.- Louis, MO, USA.
Statistical analysis
Results are presented as means±SEM. Statistical difference between before and after
the treatment with L-NAME or 7-nitroindazol was assessed by Student’s test for independent
means and p values less than 0.05 (p< 0.05) were considered statistically significant.
Statistical analyses were performed using the program
(Pharm Tools Pro, version 1.27, McCary Group Inc., PA).
Results
NSAIDs antinociception in hind paw formalin test
The i.p. administration of the different doses of piroxicam, parecoxib, dexketoprofen
or ketoprofen induced a dose-related antinociception accompanied by different potency
in both phases of the hind paw formalin test, as can be seen in [Figs. 1] and [2]. The ED50 values in mg/kg, demonstrated the following rank order of potency of the mice licking
or biting the injected paw, in the phase I: piroxicam > dexketoprofen > ketoprofen
> parecoxib and in the phase II: piroxicam > ketoprofen > parecoxib > dexketoprofen,
see [Table 1].
Fig. 1 Dose-response curves for the antinociceptive activity induced by piroxicam in phase
I (1a) and in phase II (1b) and by parecoxib in phase I (1c) and in phase II (1d) in the formalin hind paw assay. Each point is the mean with±SEM of 6–8 mice. % MPE=antinociception
evaluated as percentage of maximum possible effect.
Fig. 2 Dose-response curves for the antinociceptive activity induced by dexketoprofen in
phase I (2a) and in phase II (2b) and by ketoprofen in phase I (2c) and in phase II (2d) in the formalin hind paw assay. Each point is the mean with±SEM of 6–8 mice. % MPE=antinociception
evaluated as percentage of maximum possible effect.
Table 1 ED50 values (mean±SEM) for the antinociception induced by piroxicam, parecoxib, dexketoprofen
and ketoprofen administered i.p. in the formalin hind paw assay of mice after pretreatment
of 5 m g/kg, i.p. of L-NAME, and 5 mg/kg i.p. of 7-nitroindazol.
|
Drug
|
ED50 (mg/kg)
|
|
Phase I
|
Phase II
|
|
PIROXICAM CONTROL
|
14.53±0.70
|
8.51±0.76
|
|
PIROXICAM + L-NAME
|
6.37±1.15*
|
1.65±0.30*
|
|
PIROXICAM + 7-NITROINDAZOL
|
1.50±0.21*
|
2.85±0.17*
|
|
PARECOXIB CONTROL
|
15.81± 0.57
|
22.50±2.10
|
|
PARECOXIB + L-NAME
|
10.16±1.73*
|
5.57±0.32*
|
|
PARECOXIB + 7-NITROINDAZOL
|
3.20±0.16*
|
4.18±0.25*
|
|
DEXKETOPROFENO CONTROL
|
15.35±0.56
|
26.53±3.19
|
|
DEXKETOPROFENO + L-NAME
|
3.09±0.21*
|
6.89±0.47*
|
|
DEXKETOPROFENO + 7-NITROINDAZOL
|
3.06±0.15*
|
2.04±0.11*
|
|
kETOPROFENO CONTROL
|
15.71±0.87
|
14.61±0.46
|
|
KETOPROFENO + L-NAME
|
2.67±0.10*
|
2.10±0.47*
|
|
KETOPROFENO + 7-NITROINDAZOL
|
2.53±0.11*
|
2.15±0.15*
|
*p< 0.05 compared to the corresponding control goup. N=24.
Effect of L-NAME on the NSAIDs antinociception in hind paw formalin test
The pretreatment of mice with 5 mg/kg i.p. of L-NAME, dose with does not induce any
significant change in the basal antinociception or behavior of the mice, nevertheless,
produced a significant increase of the ED50 of both phases of piroxicam, parecoxib, dexketoprofen and ketoprofen, as shown in
[Figs. 3] and [4].
Fig. 3 ED50, in mg/kg, of piroxicam in phase I and in phase II after pretreatment of L-Name or
7-nitroindazole (3a) and parecoxib in phase I and in phase II after pretreatment of L-Name or 7-nitroindazole
(3b). Each point is the mean±SEM of 6–8 mice. * p<0.05.
Fig. 4 ED50, in mg/kg, of piroxicam in phase I and in phase II after pretreatment of L-Name or
7-nitroindazole (4a) and parecoxib in phase I and in phase II after pretreatment of L-Name or 7-nitroindazole
(4b). Each point is the mean±SEM of 6–8 mice. * p<0.05.
The order of increase of the ED50 induced by L-NAME in phase I of the NSAIDs in the
formalin test was: ketoprofen (5.88) > dexketoprofen (4.96) > piroxicam (2.28) > parecoxib
(1.55). Moreover, in phase II the order was: ketoprofen (6.95) > piroxicam (5.15)
> parecoxib (4.03) > dexketoprofen (3.85).
Effect of 7-nitroindazole on the NSAIDs antinociception in hind paw formalin test
The pretreatment of mice with 5 mg/kg i.p. of 7-nitroindazole, a dose with does not
induce any significant change in the basal antinociception or behavior of the mice,
nevertheless, produced a significant increase of the ED50 of both phases of piroxicam , parecoxib, dexketoprofen and ketoprofen, as shown in
[Figs. 3] and [4].
The order of increase of the ED50 induced by 7-nitroindazole in phase I of the NSAIDs
in the formalin test was: piroxicam (9.68), ketoprofen (6.21) > dexketoprofen (5.01)
> parecoxib (4.94). Furthermore, in phase II the order was: ketoprofen (6.79) > dexketoprofen
(6.14) > parecoxib (5.38) > piroxicam (2.98).
Discussion
The present study was performed to determine the antinociceptive and anti-inflammatory
activities of four NSAIDs using the hind paw formalin test and if the effects induced
by L-NAME and 7-nitroindazole are involved in the activity of NSAIDs The results demonstrated
that piroxicam, parecoxib, dexketoprofen and ketoprofen are able to inhibit both phases
of the hind paw formalin test.
The findings of this work are in agreement with the typical biphasic response reported
previously [2]
[3]
[15]. Phase I results from direct stimulation of nociceptors, while phase II involves
a period of sensitization, during which inflammatory phenomena occur through peripheral
mechanisms [1]
[2]. Though, the results obtained in the present study, differ from previously reported,
in that the administration of formalin in this test only produces one phase, being
it phase II specifically [16]
[17].
In the present work, pretreatment of mice with L-NAME resulted in a significant increase
in the antinociceptive potency of each NSAID, reflected by a significant decrease
in ED50, both in phase I and in phase II. This nociceptive increase is consistent with similar
effects of L-NAME in diazepam, clonazepam, chlordiazepoxide, tadalafil [18]
[19]. However, this nociceptive increase is not consistent with the decrease in the antinociception
induced by L-NAME in modafinil, ketamine, diclofenac, meloxicam and carbamazepine
[19]
[20]
[21]
[22]
[23].
Pretreatment of mice with 7-nitroindazole increased the antinociceptive potency of
the NSAIDs used in this study, with the consequent significant decrease in ED50, in both phases of the formalin test. This finding is in agreement with a similar
effect induced by 7-nitroindazole in diazepam clonazepam,. chlordiazepoxide and tapentadol
analgesia [18]
[24]. However, the effect of produced by 7-nitroindazole is not consistent with the reported
for modafinil and dexmedetomidine [19]
[25]. The reasons for these discrepancies could be explained by the different strains
of mice, the period of work of the protocol, the different doses used and others.
The significative decrease of the ED50 of the different NSAIDs produced by L-NAME is related to its COXs inhibitory capacity.
Phase I is headed by ketoprofen, a so-called non-selective NSAID, followed by dexketoprofen
and piroxicam, recognized as COX-1 inhibitors and finally, a COX-2 inhibitor, parecoxib.
7-nitroindazole, in this phase, decrease the ED50 of piroxicam, first, then to ketoprofen and dexketoprofen and ends with parecoxib.
The increase in NSAID potency, expressed by the decrease in its ED50, in the case
of L-NAME, follows the following course in phase II: ketoprofen, then piroxicam, parecoxib
and finally dexketoprofen. In relation to the effect of 7-nitroindazole, the decrease
in DE50 is initiated by ketoprofen, followed by dexketoprofen and parecoxib, ending
with piroxicam. In summary, the increase in antinociceptive potency, evaluated by
a significant decrease in the ED50 of NSAIDs, produced by both L-NAME and 7-nitroindazole, has a higher affinity for
COX-1 inhibitors than for COX-2 inhibitors.
The increase in the potency of NSAIDs, reported in this work, by the action of NOS
inhibitors, seems to be due to the effect on each of the components. So, first the
effectiveness of the non-selective inhibitor NOS, L-NAME and the selective nNOS inhibitor,
7-NI in increase the power of piroxicam, parecoxib, dexketoprofen and ketoprofen indicates
that NO activity is involved in both phases of the hind paw formalin test. It is know,
that after formalin administration, induces the release of substance P and glutamate
and finally the release of NO that provokes central sensitization [19]
[24].Furthermore, studies with NOS inhibitors and cyclooxygenase inhibitor, suggest that
NO stimulates the activity of COX. nNOS and L-NAME has been involved in the spinal
transmission of nociception in animal models of acute and chronic pain [7]
[11]
[13]
[14]
[26]
[27]. On the other hand, the results obtained in this study, demonstrated that NO was
involved in the mechanism of antinociception of piroxicam, parecoxib, dexketoprofen
and ketoprofen in both phases of the hind paw formalin test. The mechanism of this
antinociceptive action is similar to that previously described for baclofen, morphine,
dipyrone, diclofenac, acetylcholine, bremazocine, xylazine and others [21]
[22]
[28].
In the present study, a significant increase in the potency of some NSAIDs is reported
due to NOS inhibitors. However, it is suggested that in addition to the COXs-NO interaction,
other mechanisms of action that have been attributed to NSAIDs and that could contribute
to the findings described should be taken into account. Among them are: interaction
with endocannabinoids, monoaminergic and cholinergic systems [29]. In addition, others such as lactoferrin modulation; inhibition of prostaglandin
keto reductase; phospholipase action; negative regulation of L-selectin; TNF-α or
IL-1β inhibition [30].
Conclusions
In this study the intraperitoneal administration of piroxicam, parecoxib, dexketoprofen
and ketoprofen produced antinociception in both phases of the mice formalin hind paw
assay. The pretreatment of the mice with L-NAME or 7-nitroindazol induced a significant
increase of the analgesic power of the NSAIDs, with a significative reduction of the
ED50. It is suggested that NO may be involved in both phases of the trial, which means
that nitric oxide regulates the bioactivity of NSAIDs.
Author Contributions
All the authors contributed equally in this article.
