Evidence for a New Mechanism of Action of Diclofenac: Activation of ...

Proc. West. Pharmacol. Soc. 44: 19-21 (2001)
Evidence for a New Mechanism of Action of Diclofenac: Activation of K + Channels
MARIO I. ORTIZ 1,2 , GILBERTO CASTAÑEDA-HERNÁNDEZ 1 , RODOLFO ROSAS 3 ,
GUADALUPE C. VIDAL-CANTÚ 4 & VINICIO GRANADOS-SOTO 4*
1 Sección Externa de Farmacología, Centro de Investigación y de Estudios Avanzados del IPN, México, D.F., Mexico; 2 Área Académica de
Medicina del Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Mexico; 3 Novartis Farmacéutica, México, D.F.,
Mexico; 4 Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados del IPN, Czda. de los Tenorios 235, Col.
Granjas Coapa, 14330 México, D.F., Mexico
Diclofenac is a non-steroidal anti-inflammatory drug
(NSAID) with strong anti-inflammatory, antipyretic and
analgesic activity [1,2]. This drug has shown to be effective
in the treatment of rheumatic and non-rheumatic conditions
[2]. Diclofenac has been considered a NSAID
since its introduction [3]. However, it has been suggested
that, in addition to the in vitro and in vivo inhibition of
COX [4,5], diclofenac apparently possesses additional
mechanisms of action [6]. Some reports have suggested a
central analgesic action of diclofenac mediated by endogenous
opioids in brain stem nuclei related to the control
of nociception [7-10]. Notwithstanding, there is evidence
that naloxone (an opioid antagonist) or N-methylnalorphine
(a peripheral opioid antagonist) can block the
antinociception produced by morphine, but not that produced
by diclofenac [11]. These results demonstrate that
diclofenac action is not due to peripheral or central release
of an opioid-like substance.
On the other hand, it has been demonstrated that the
inhibition of serotonergic transmission by pretreatment
with methiothepin, ritanserin, parachlorophenylalanine or
5,7-dihydroxytryptamine reduces the antinociceptive effect
of diclofenac [10], suggesting a direct relationship
between central serotonergic mechanisms and diclofenacinduced
antinociception. In addition, diclofenac is able to
block in a dose-dependent manner the hyperalgesia induced
by intrathecal NMDA, but not that induced by intrathecal
substance P or AMPA. This effect is reversed by
L-arginine, but not by D-arginine. These results suggest
that diclofenac is able to interfere with the pronociceptive
activity of the nitric oxide system at the spinal level [10].
It has also been demonstrated that the antinociceptive
effect of diclofenac can be blocked by peripheral administration
of either NO or cyclic GMP synthesis inhibitors,
suggesting that at least part of its antinociceptive effect is
through the activation of the NO-cyclic GMP pathway in
the periphery [11,12]. Other reports have also found that
ketorolac, metamizol and meloxicam have similar actions
[13-15]. Recently we have observed that glibenclamide
(an ATP-sensitive K + channel inhibitor) is able to block
the peripheral antinociceptive effect of ketorolac in the rat
(unpublished observation). Therefore, in this work we
tested the participation of the ATP-sensitive K + channel in
the antinociceptive effect of diclofenac in the formalin
test.
METHODS: Female Wistar rats (weight range, 160-180 g) from our
breeding facilities were used. All experiments followed the Guidelines
on Ethical Standards for Investigation of Experimental Pain in Animals
[16]. Additionally, the study was approved by the Institutional Animal
Care Committee.
Evaluation of antinociceptive activity. Antinociception was assessed
by the formalin test. Rats were placed in a open Plexiglas observation
chamber for 30 min to allow them to accommodate to their
surroundings, then they were removed for formalin administration.
Fifty µl of dilute formalin (1%) were injected into the dorsal surface of
the right hind paw. The animal was then returned to the chamber for
observation. A mirror was placed behind the chamber to enable unhindered
observation of the formalin-injected paw. Nociceptive behavior
was quantified as the number of flinches of the injected paw during 1
min-periods every 5 min up to 60 min after injection as described previously
[15]. The flinching was readily discerned and was characterized
as rapid and brief withdrawal or flexing the injected paw. At the
end of the experiment the rats were killed in a CO 2 chamber.
Drugs. Diclofenac sodium was a gift of Merck Mexico (Mexico
City). Glibenclamide and 4-aminopyridine (4-AP) were purchased
from Sigma (St. Louis, MO, USA). Other substances were of analytical
grade.
Study design. Female Wistar rats received vehicle or increasing
doses of diclofenac (50-200 µg/paw) 20 min before formalin injection.
To assess if the antinociceptive effect was due to a local action, formalin
was administered in one paw and the tested drug in the contralateral
paw. In other groups, rats received diclofenac (100 µg) and either
glibenclamide (10-50 µg) or 4-AP (10-50 µg). Doses of drugs were
based on pilot studies in our model. Rats in all groups were tested for
possible side effects such as reduction of righting, stepping, corneal
and pinna reflexes and catalepsy before and after drug administration.
Data analysis and statistics. Results are presented as means ±
SEM for 6-8 animals per group. Curves were made for number of
flinches against time. Data are expressed as the area under the number
of flinches against time curve (AUC). Analysis of variance followed by
the Tukey´s test was used to test differences between treatments. A
value of p < 0.05 was considered to be significant.
RESULTS: Formalin administration produced a typical
pattern of flinching behavior. The first phase started immediately
after administration of formalin and diminished
gradually in about 10 min. The second phase started at
about 15 min and lasted for 1 h [15]. Ipsilateral, but not
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contralateral, local administration of diclofenac produced
a dose-dependent reduction in the flinching behavior otherwise
observed after formalin injection (Fig. 1). Diclofenac
significantly reduced the number of flinches during
phase two (p < 0.05), but not during phase one. No
side effects were observed in either group, control or
treated.
It was observed that glibenclamide, an ATP-sensitive
K + channel blocker, was able to diminish diclofenac-
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induced antinociception. The fact that glibenclamide specifically
blocks ATP-sensitive K + channels, with no effect
on Ca 2+ - or voltage-dependent K + channels [18-20] suggests
that diclofenac produced, at least part of, its antinociceptive
effect through the opening of the ATP-sensitive
K + channel. To our knowledge, this is the first report on
the participation of the ATP-sensitive K + channel in the
antinociceptive activity of a NSAID. However, 4-AP also
blocked diclofenac-induced antinociception. As 4-AP is
considered as a voltage-dependent K + channel inhibitor
[21], our results suggest that, beside the ATP-sensitive K +
channels, diclofenac also opens the voltage-dependent K +
channel.
Figure 1. Local antinociceptive effect of diclofenac during the second
phase of the formalin test. Rats received intraplantar pretreatment with
saline and diclofenac and then a formalin (1%) injection (50 µl). Data
are expressed as the area under the number of flinches against time
curve (AUC). Bars are the means ± SEM of 6-8 animals. *Significantly
different from saline (p < 0.05), as determined by analysis of variance
followed by the Tukey´s test.
Glibenclamide or 4-AP (10-50 µg/paw) did not produce
any effect by themselves. However, either glibenclamide
or 4-AP (ATP-sensitive and voltage-dependent
K + channel inhibitors, respectively), but not vehicle, significantly
blocked diclofenac-induced antinociception
during the second phase of the formalin test (Fig. 2). In no
group were visible side effects observed.
DISCUSSION: In this study we observed peripheral
antinociception with diclofenac. The peripheral effect of
diclofenac was due to a local action as the administration
of this drug at the contralateral paw was ineffective. It is
well known that diclofenac is a non-selective COX inhibitor
[17]. Therefore it is likely that its antinociceptive effect
could result from COX inhibition. However, other
actions [11] different from COX inhibition cannot be
ruled out.
Figure 2. Effect of glibenclamide (Gli) and 4-aminopirydine (4-AP) on
the antinociceptive activity of diclofenac in the formalin test. Rats
received intraplantar pretreatment with diclofenac (100 µg) alone or
coadministered with Gli or 4-AP and then a formalin (1%) injection
(50 µl). Data are expressed as the area under the number of flinches
against time curve (AUC). Bars are the mean ± SEM of 6-8 animals. *
Significantly different from vehicle (p < 0.05); # significantly different
from the diclofenac group, as determined by analysis of variance followed
by the Tukey´s test.
In summary, diclofenac produced peripheral antinociception
in the formalin test in the rat. The antinociceptive
effect of diclofenac was antagonized by glibenclamide
and 4-AP. These results strongly suggest that, besides
the inhibitory action on prostaglandin synthesis, the
opening of the ATP-sensitive and voltage-dependent K +
channels has an important role in the peripheral antinociception
of diclofenac in the formalin test.
ACKNOWLEDGEMENTS: M.I. Ortiz is a PROMEP
fellow. Authors greatly appreciate the bibliographic assistance
of H. Vázquez.
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