Full text of DOI:10.1038/sj.bjp.0704589

British Journal of Pharmacology (2002) 135, 1556 ± 1562
ã
2002 Nature Publishing Group All rights reserved 0007 ± 1188/02 $25.00
www.nature.com/bjp
NCX-701 (nitroparacetamol) is an e€ective antinociceptive agent
in rat withdrawal re¯exes and wind-up
1
2
1E. Alfonso Romero-Sandoval, Javier Mazario, David Howat & *,1Juan F. Herrero
2
1Departamento de Fisiologõa, Facultad de Medicina, Universidad de Alcala, Madrid, Spain and NicOx S.A., Sophia-Antipolis,
France
1
Non-steroidal anti-in¯ammatory drugs (NSAIDs) are e€ective anti-in¯ammatory and analgesic
drugs although they also induce unwanted side e€ects due to the inhibition of the physiological
e€ects regulated by prostaglandins. This has led to the search for new compounds with fewer side
e€ects, such as the nitro-NSAIDs (NO-NSAIDs). Paracetamol is an analgesic drug devoid of some
of the side e€ect of the NSAIDs but without anti-in¯ammatory activity. NCX-701 is a nitric oxide
releasing version of paracetamol with anti-in¯ammatory and analgesic properties.
2
We have tested, in the single motor unit technique, the antinociceptive actions of intravenous
cumulative doses of NCX-701 vs paracetamol, studying their antinociceptive e€ects in responses to
noxious mechanical and electrical stimulation (wind-up).
3
Paracetamol did not induce any signi®cant e€ect at the doses tested (maximum of
480 mmol kg71, 72.5 mg kg71). NCX-701 however was very e€ective in reducing responses to
noxious mechanical stimulation (32+10% of control response) and wind-up (ED50 of
147+1 mmol kg71, 41.5+0.3 mg kg71). The inhibition was not reversed by 1 mg kg71 of the opioid
antagonist naloxone. In control experiments performed with either the vehicle or the NO donor
NOC-18, no signi®cant changes were observed in the nociceptive responses studied.
4
We conclude that NCX-701 is a very e€ective non-opioid antinociceptive agent in normal
animals and its action is located mainly at central areas. The antinociceptive e€ect was not due
solely to the release of NO.
British Journal of Pharmacology (2002) 135, 1556 ± 1562
Keywords: Pain; analgesia; spinal cord; non-steroidal anti-in¯ammatory drugs; paracetamol
Abbreviations: ANOVA, analysis of variance; CNS, central nervous system; COX, cyclo-oxygenase; DMSO, dimethyl
sulphoxide; ED50, e€ective dose 50; MAP, mean arterial pressure; MED, minimum e€ective dose; NMDA, N-
Methyl-D-aspartate; NO, nitric oxide; NOC-18 (DETA NONOate), (Z)-1-[2(2-Aminoethyl)-N-(2-ammonio-
ethyl)amino]diazen-1-ium-1,2-diolate; NO-NSAID, nitro non-steroidal anti-in¯ammatory drug; NSAID, non-
steroidal anti-in¯ammatory drug; PGE2, prostaglandin E2; SMU, single motor unit
Introduction
Unwanted side e€ects induced by non-steroidal anti-in¯am-
matory drugs (NSAIDs) led to the search for new molecules
with e€ective anti-in¯ammatory and analgesic actions but
devoid of the unsafe e€ects. Nitro-NSAIDs (NO-NSAIDs)
are a group of new NSAIDs developed as a result of that
strategy (Del Soldato et al., 1999). Since nitric oxide (NO)
has cytoprotective properties in the gastric mucosa similar to
those mediated by prostaglandins, some classic NSAIDs
(naproxen, diclofenac, ¯urbiprofen and aspirin for example)
have been modi®ed to include in their structure NO donor
molecules, in an attempt to counterbalance the gastrointest-
inal damage and other side e€ects induced by the action of
the NSAIDs. As a result, NO-NSAIDs seem to be safer to
the grastrointestinal mucosa than are classic NSAIDs
(Wallace et al., 1999), more e€ective anti-in¯ammatory drugs
(Fiorucci, 2001) and more potent analgesic than their parent
compounds (Al-Swayeh et al., 2000). This type of `latest
generation' NSAIDs might open new perspectives in the
treatment of chronic pain, especially in those patients at risk
of developing severe complications. In the present study we
have studied the analgesic e€ects of a new nitric oxide
releasing version of paracetamol NCX-701, using the single
motor unit (SMU) technique which has been shown to be a
good model to test analgesics in acute experiments (Mazario
et al., 2001). We have compared the analgesic actions of
NCX-701 vs paracetamol and have also studied their
e€ectiveness in reducing responses to noxious mechanical
(natural) and electrical stimulation (wind-up), and therefore
compared peripheral vs central actions of these drugs
(Herrero et al., 2000). Preliminary results have been
published elsewhere in abstract format (Romero-Sandoval
et al., 2001).
Methods
Animals, preparatory surgery and groups of experiments
*Author for correspondence at: Departamento de Fisiologõa, Campus
Universitario, Universidad de Alcala, Alcala de Henares, 28871
Madrid, Spain; E-mail: juanf.herrero@uah.es
The experiments were performed in 21 male Wistar rats
weighing 235 ± 350 g divided into two groups: animals treated

E.A. Romero-Sandoval et al
Antinociception induced by NCX-701
1557
with paracetamol (n=6) and animals treated with nitro-
paracetamol (NCX-701, n=9). Another two groups of
control experiments (n=3 each) were performed to test the
possible e€ect of the vehicle used and the actions of the NO
donor, NOC-18 (DETA NONOate, Alexis), with similar NO
release rate to that of NCX-701 (see Results). The SMU
technique has been described in detail elsewhere (Herrero &
Headley, 1991; Solano & Herrero, 1997). The preparatory
surgery consisted in the cannulation of the trachea, one
carotid artery to register the blood pressure, and two
super®cial jugular veins, and was performed under halothane
anaesthesia (5% in oxygen for induction and 2 ± 3% for
maintenance). After the surgery, halothane was discontinued
and the anaesthesia continued with alpha-chloralose (Sigma,
50 mg kg71 initial dose and 30 mg kg71 h71 by perfusion
pump for maintenance in a rate of 1 ml h71, diluted in
saline). The right hind limb was ®xed in inframaximal
extension in a Perspex block using plaster of Paris (Figure
1). The core temperature was maintained at 37+0.58C by
means of a feed-back controlled heating blanket and the
blood pressure was continuously monitored. The preparation
was left to rest for at least an 1 h before the experiment
started.
noxious mechanical and electrical stimuli applied in 3 min
cycles. Figure 1 illustrates the preparation and shows an
original recording of
a SMU during two cycles of
stimulation. Paracetamol or NCX-701 were tested only when
the responses observed with either stimulus were stable.
Noxious mechanical stimulation was applied over an area of
14 mm2 using a computer controlled pincher, and a force of
200 mN over the threshold intensity, threshold being the
minimum pressure required to evoke a constant ®ring rate for
at least 10 s of stimulation. The electrical stimulation was
applied using two 0.2 mm needles inserted percutaneously in
the most sensitive area of the cutaneous receptive ®eld, with
16 pulses of 2 ms width, 1 Hz and an intensity of twice the
threshold current for long latency responses (C-®bre
responses, Herrero & Cervero, 1996a). Only units with a
stable ®ring rate and summation of responses to constant
intensity repetitive electrical stimulation (wind-up) were
selected for the experiments (Figure 1; Herrero et al., 2000).
Figure 1 shows an original recording of a single unit with a
constant ®ring rate after noxious mechanical stimulation and
a progressive increase in responses to high intensity electrical
stimulation.
Drugs and collection and analysis of data
Stimulus presentation and recording systems
Paracetamol and NCX-701 (kindly supplied by NICOX S.A.)
were dissolved in DMSO (Sigma) and polyethylene glycol
(1 : 1) (Panreac) in a concentration of 50 mM, diluted in saline
and administered intravenously in cumulative log2 regime.
Single motor units (SMUs) were recorded by means of
bipolar tungsten electrodes inserted percutaneously into
muscles of the right hind limb, and were activated by
Figure 1 Methods and protocol of stimulation. (A) Single motor units were recorded by means of bipolar tungsten electrodes
inserted percutaneously in muscles of the hind limb. (B) Responses were elicited in three min cycles alternating 10 s noxious
mechanical stimulation (200 mN above response threshold) and high intensity repetitive electrical stimulation (16 pulses, 1 Hz, 2 ms
width, twice times threshold intensity for C-®bre activation). Blood pressure was continuously monitored. (C) Repetitive electrical
stimulation induced in all cases a progressive increment of the number of responses (wind-up). These responses were studied as the
total number of spikes recorded between 150 and 650 ms after each stimulus.
British Journal of Pharmacology vol 135 (6)

1558
E.A. Romero-Sandoval et al
Antinociception induced by NCX-701
The initial dose used was 15 mmol kg71 and the highest dose
used was 480 mmol kg71. Each dose was administered every
seven cycles of stimulation (21 min). In three experiments, the
e€ect of NCX-701 was challenged with 1 mg kg71 of the
opioid antagonist naloxone (Sigma), administered 27 min
after the last dose tested. The animals were killed at the end
of the experiments with an overdose of sodium pentobarbi-
tone (Euta Lender, Normon). The number of spikes counted
in the last two cycles between each dose were averaged and
the mean compared to the control response, control being the
mean of the three responses previous to the administration of
the ®rst dose. The data from the electrical stimulation were
analysed by counting the number of spikes evoked between
150 and 650 ms after each stimulus (C-®bre responses,
Herrero & Cervero, 1996a).
The collection of data and stimulation protocols were
performed by computer using commercial software (CED,
UK; Spike 2). Statistical analyses were performed using the
one-way analysis of variance (ANOVA) with post-hoc
Dunnett's test for comparisons between responses observed
after the administration of each dose and control. Compar-
ison between responses of paracetamol and NCX-701 was
performed using the non-parametric Mann-Whitney U-test.
Statistical comparisons were performed using commercial
software (GraphPad-Prism and GraphPad-Instat for Win-
dows). The data are presented as the mean+s.e.m. All the
experiments performed in this study were carried out in
accordance with European Union legislation regarding the
use of animals for experimental protocols.
Results
Effects of paracetamol and NCX-701 on responses to
noxious mechanical stimulation
Figure 2 shows the e€ect induced by cumulative doses of
paracetamol and NCX-701 in original SMU recordings.
Pooled data on responses to noxious mechanical stimulation
are represented in Figure 3. The administration of para-
cetamol did not induce any signi®cant change in responses to
noxious mechanical stimulation at the doses tested (117+5%
control with the maximal dose used of 480 mmol kg71
,
72.5 mg kg71, n=6). NCX-701, however, induced a dose-
dependent depression of these responses with a minimum
signi®cant e€ective dose (MED) of 120 mmol kg71 (P50.05).
The maximum e€ect observed was 32+10% of control
(P50.01, n=9), and the ED50 was of 147+1.1 mmol kg71
(41.5+0.3 mg kg71). The number of spikes evoked by the
noxious mechanical stimulation used was very similar in all
experiments, with a ®ring rate of around 30 Hz (24 ± 32 Hz).
The e€ect of the drugs was not signi®cantly di€erent when
comparison between di€erent responses was studied. To
check whether or not this e€ect was mediated by opioid
receptors, the antinociceptive e€ect of NCX-701 was
challenged in three experiments with the non-selective opioid
antagonist naloxone at a dose of 1 mg kg71 (Figure 3). No
recovery of the e€ect induced by NCX-701 was observed in
any of the tests performed (13+6% control response,
P50.01, n=3).
Figure 2 Original recordings of two di€erent single motor units previous to and after the administration of NCX-701 (A) and
paracetamol (B). NCX-701 induced a potent dose-dependent reduction of responses to either noxious mechanical stimulation or
repetitive electrical stimulation. Paracetamol however did not induce any signi®cant reduction of responses to either type of
stimulation.
British Journal of Pharmacology vol 135 (6)

E.A. Romero-Sandoval et al
Antinociception induced by NCX-701
1559
Figure
4
illustrates the responses observed after the
This increase in the number of responses was not signi®cant
and was not dose-dependent.
administration of equivalent doses of the vehicle used to
dissolve either paracetamol or NCX-701. The administration
of the vehicle did not induce any signi®cant reduction of
responses to noxious mechanical stimulation. In fact, the
responses were increased up to a maximum of 140.7+18%
Effects of paracetamol and NCX-701 on responses to
repetitive electrical stimulation
(n=3) of control response with the dose of 120 mmol kg71
.
High intensity electrical stimulation induced an obvious
wind-up in all the experiments performed (Figure 5) and
the responses elicited with the ®rst pulse were similar in all
curves. The administration of cumulative doses of para-
cetamol did not induce any signi®cant reduction on wind-up
(Figure 5), although a non-signi®cant increase of responses
evoked with the ®rst pulse was observed. The administration
of NCX-701 however induced a very e€ective reduction of
wind-up without changing the number of spikes recorded
with the ®rst pulse applied. As in responses to noxious
mechanical stimulation, the e€ect was dose-dependent with a
Figure 3 Pooled data of SMU responses evoked by noxious
mechanical stimulation after the administration of cumulative doses
of paracetamol and NCX-701. The administration of paracetamol
did not induce any signi®cant e€ect. NCX-701, however, induced a
dose-dependent reduction of responses, with an ED50 of
147+1.1 mmol kg71 (41.5+0.3 mg kg71). The e€ect was challenged
in three experiments with 1 mg kg71 of the opioid antagonist
naloxone but no recovery was observed. (*P50.05; **P50.01,
comparison vs control response using the one-way ANOVA, with the
post-hoc Dunnett test; #P50.05; ##P50.01, comparison between
paracetamol and NCX-701 using the non-parametric Mann-Whitney
U-test.)
Figure 5 SMU wind-up observed after the administration of
cumulative doses of paracetamol and NCX-701. The administration
of paracetamol did not induce any signi®cant e€ect of wind-up
responses. NCX-701 induced a dose-dependent reduction of wind-up
with a MED of 240 mmol kg71. As for responses to mechanical
stimulation, the e€ect was not reversed by 1 mg kg71 of the opioid
antagonist naloxone. (Statistical comparison between responses
observed with each dose and the control response was made using
the one-way analysis of variance, ANOVA, with the post-hoc
Dunnett test, ***P50.001.)
Figure 4 Pooled data of SMU responses evoked by noxious
mechanical and electrical stimulation after the administration of
equivalent doses of the vehicle used. The administration of the
vehicle induced a non dose-dependent and not signi®cant increase of
responses to noxious mechanical and electrical stimulation.
British Journal of Pharmacology vol 135 (6)

1560
E.A. Romero-Sandoval et al
Antinociception induced by NCX-701
MED of 240 mmol kg71 (P50.001). The administration of
1 mg kg71 of naloxone did not reverse the depression
induced by NCX-701 (Figure 5, P50.001).
The administration of equivalent doses of the vehicle used
induced an increase in the spikes counted, up to a maximum
of 173.8+10% (n=3) of control response, at a dose of
240 mmol kg71 (Figure 4). This increase was not dose-
dependent.
480 mmol kg71, n=3). This e€ect was neither dose-dependent
nor statistically signi®cant.
The e€ect of NOC-18 on blood pressure was also analysed
and, as shown in Figure 7, no signi®cant reduction in MAP
was observed after the administration of any of the doses
studied.
Discussion
Effects of paracetamol and NCX-701 on blood pressure
The main observation made in this study is that NCX-701 is an
e€ective antinociceptive agent in normal, non-in¯amed
animals. Paracetamol, like other NSAIDs, is usually not
e€ective in experiments performed in the normal, non-in¯amed
situation, since the synthesis of prostaglandins is not
augmented and therefore there is not a clear target to be
modulated. In similar experiments, we have previously
observed no e€ect caused on SMU responses to noxious
mechanical and electrical stimulation, for instance, by ¯unixin
(Herrero et al., 1996) or ¯urbiprofen (Mazario et al., 2001). An
e€ective analgesia can be observed however if the compound
Blood pressure was monitored throughout the experiment
and, as for responses to noxious mechanical and electrical
stimulation, data analysed in the last two cycles of
stimulation between each dose were averaged and the mean
compared to the control response. Figure 6 shows pooled
data for mean arterial pressure (MAP) values observed after
the administration of paracetamol and NCX-701. As can be
seen, no signi®cant change in MAP was observed after the
administration of either drug.
Control experiments performed with NOC-18
(DETA NONOate)
In order to assess whether the antinociceptive e€ects observed
with NCX-701 were only due to the release of NO, three
more experiments were performed using the NO donor NOC-
18. This compound was chosen since its ability to release NO
is very slow (Hrabie et al., 1993; Keefer et al., 1996) similar
to that of NCX-701 (Del Soldato et al., 1999; Futter et al.,
2001). Figure
7 shows the results observed after the
administration of equivalent doses of NOC-18 in responses
to noxious mechanical and electrical stimulation. A small
reduction in the number of spikes was observed either in
responses to noxious mechanical stimulation (maximum of
75.1+2% of control response with 30 mmol kg71, n=3) or
electrical stimulation (maximum of 56.8+2% with
Figure 7 E€ect of NOC-18 on mean arterial pressure and SMU
responses evoked by noxious mechanical and electrical stimulation.
(A) The administration of cumulative doses of the NO donor NOC-18
did not induce any signi®cant change in SMU responses to noxious
mechanical and electrical stimulation. (B) NOC-18 did not either
modify the mean arterial pressure values in a signi®cant manner.
Figure 6 Pooled data of mean arterial pressure values previous to
and after the administration of cumulative doses of paracetamol and
NCX-701. Neither of the drugs studied induced any signi®cant
change in blood pressure (data taken and averaged 18 and 21 min
after the administration of each dose).
British Journal of Pharmacology vol 135 (6)

E.A. Romero-Sandoval et al
Antinociception induced by NCX-701
1561
crosses the blood-brain barrier easily, and an action upon the
central processing of nociceptive information is directly exerted
(Mazario & Herrero, 1999; Mazario et al., 2001). Paracetamol
has been shown to have central antinociceptive e€ects, but only
when administered at doses much higher that those used in the
present study (Pelissier et al., 1995; Pini et al., 1997). In our
hands, a central action of paracetamol does not seem very
important when studied in the present conditions, since a
central e€ect would be expected to modify the excitability of
SMUs and the wind-up phenomenon, at least partially
(Herrero et al., 2000, see below). NCX-701 however was very
e€ective either in responses to noxious mechanical stimulation
or high intensity electrical stimulation. An action at central
sites is supported by the strong e€ect exerted on the wind-up
phenomenon. In this phenomenon, repetitive electrical stimu-
lation induces a progressive increase of nociceptive responses
from spinal cord neurones (Herrero et al., 2000, and references
within) and is mediated by NMDA (Davies & Lodge, 1987;
Dickenson & Sullivan, 1987) and NK1 receptors (De Felipe et
al., 1998). A reduction of wind-up implies an inhibitory action
of the circuitry involved in its generation, which is located in
the central nervous system, at spinal cord level (see Herrero et
al., 2000 for further discussion), although a modulation of the
system by higher levels in the CNS is also possible (Herrero &
Cervero, 1996b). The e€ect of NCX-701 therefore takes place
presumably at central sites rather than in the periphery. A
second possibility is that NCX-701 induced an increase in the
threshold for activation of nociceptive a€erents, and therefore
a reduction in the amount of input reaching the spinal cord, as
described with some opioid agonists (Chapman & Dickenson,
1992). This is not probable since the experiments showed that
the number of spikes recorded with the ®rst electrical stimulus
was very similar at all doses tested.
agonist fentanyl (Thorn et al., 1994; Gaitan & Herrero,
2002). Since no reversal of the e€ect was observed, we
conclude that the e€ect observed with NCX-701 was not due,
in our experiments, to an action on opioid receptors.
Another possibility is that NO induced an antinociceptive
e€ect in its own. This, again, does not seem to be the case
since the experiments performed with NOC-18 showed no
variation in the responses evoked by either noxious
mechanical or electrical stimulation. Also, the possibility that
a general depression of the system due to a blood pressure
fall, induced by a NO-mediated vasodilation, is not likely
since the release of NO by NCX-701 is very slow and, similar
to that observed with NOC-18, no major changes were
observed in blood pressure with the doses studied.
It is therefore possible that the progressive release of NO
favoured or promoted an antinociceptive e€ect of para-
cetamol (and/or vice versa), perhaps by a di€erent mechan-
ism of action. In fact, although we have not seen any clear
e€ect after the administration of NOC-18, NO donors may
induce either pronociception or antinociception, and the most
clear e€ect in spinal cord-mediated withdrawal re¯exes seems
to be antinociceptive (Sousa & Prado, 2001). This action
however appears to be NO concentration-dependent (Kur-
ihara & Yoshioka, 1996; Sousa & Prado, 2001). Also, NO
synthesis in the CNS is mediated by the constitutive neuronal
isoform of the NO synthase which seems to be positively
modulated by the activation of NMDA (Kiss & Vizi, 2001).
It has been suggested that NO release, as a consequence of
NMDA receptor activation, modulates negatively in turn the
release of glutamate, by enhancing the amount of mono-
amines in the synaptic space (Kiss & Vizi, 2001). On the
other hand, PGE2 enhances the release of glutamate and
aspartate in the spinal cord (Nishihara et al., 1995) and the
blockade of COX activity might reduce the levels of PGE2
and therefore the release of glutamate. If this is true, NCX-
701 might negatively modulate the activity of NMDA
receptors by a joint action of the NO released and the
paracetamol molecule, depressing the release of glutamate
through di€erent mechanisms, and causing an e€ect greater
than that observed when administered separately. This
possibility would explain the potent e€ect of NCX-701
observed in the present study in wind-up, a phenomenon
mediated by NMDA receptors (see above). Nevertheless,
further experiments are needed to assess this possibility.
In conclusion, NCX-701 is a potent and e€ective analgesic
agent in normal animals after intravenous administration.
The e€ect is not due solely to the action of NO and, at least
in part, is mediated at central sites, probably the spinal cord,
since wind-up was greatly inhibited.
The question that remains unanswered is how the NO
released by NCX-701 was able to enhance the antinociceptive
e€ects of paracetamol. A possibility is that NO was somehow
able to facilitate the entrance of paracetamol into the CNS;
this might explain why NCX-701 inhibits wind-up whereas
paracetamol does not. Although possible, it does not seem
probable, since paracetamol crosses the blood-brain barrier
easily (see for instance Ochs et al., 1985; Bannwarth et al.,
1989). Also, NO-NSAIDs have been suggested to have COX-
independent activities (Del Soldato et al., 1999; Fiorucci,
2001; Kiss & Vizi, 2001) and this may also be true for NCX-
701. In fact, in similar experiments to those performed in the
present study, we and others have observed that some
NSAIDs, including paracetamol, may induce
a potent
antinociceptive action by modulating opioid systems (Herrero
& Headley, 1996; Pini et al., 1997). It is for that reason that
we challenged the inhibition of nociceptive responses induced
by NCX-701 with high doses of the opioid antagonist
naloxone. The dose of naloxone used is several times higher
than that needed in similar experiments to reverse a total
inhibition of responses induced by morphine (Herrero &
Headley, 1991) or to prevent any action of the m-opioid
This work was supported by NicOx S.A. and by the Comunidad de
Madrid, grant 08.5/0068/2000. E. Alfonso Romero-Sandoval is a
fellow of the AECI (MUTIS program); Javier Mazario is a fellow
of the Spanish Ministry of Education (MECD). We thank Mr K.
Robinson for his help with the English revision.
References
AL-SWAYEH, O.A., CLIFFORD, R.H., DEL SOLDATO, P. & MOORE,
P.K. (2000). A comparison of the anti-in¯ammatory and anti-
nociceptive activity of nitroaspirin and aspirin. Br. J. Pharma-
col., 129, 343 ± 350.
BANNWARTH, B., NETTER, P., POUREL, J., ROYER, R.J. &
GAUCHER, A. (1989). Clinical pharmacokinetics of nonsteroidal
anti-in¯ammatory drugs in cerebrospinal ¯uid. Biomed. Phar-
macother., 43, 121 ± 126.
British Journal of Pharmacology vol 135 (6)

1562
E.A. Romero-Sandoval et al
Antinociception induced by NCX-701
CHAPMAN, V. & DICKENSON, A.H. (1992). The combination of
NMDA antagonism and morphine produces profound antinoci-
ception in the rat dorsal horn. Brain Res., 573, 321 ± 323.
DAVIES, S.N. & LODGE, D. (1987). Evidence for the involvement of
N-methylaspartate receptors in `wind-up' of class 2 neurones in
the dorsal horn of the rat. Brain Res., 424, 402 ± 406.
KEEFER, L.K., NIMS, R.W., DAVIES, K.M. & WINK, D.A. (1996).
``NONOates'' (1-substituted diazen-1-ium-1,2.diolates) as nitric
oxide donors: convenient nitric oxide dosage forms. Methods
Enzymol., 268, 281 ± 293.
KISS, J.P. & VIZI, E.S. (2001). Nitric oxide: a novel link between
synaptic and nonsynaptic transmission. TiNS, 24, 211 ± 215.
DE FELIPE, C., HERRERO, J.F., O'BRIENY, J.A. PALMERY, J.A.,
DOYLEY, C.A., SMITH, A.J.H., LAIRD, J.M.A., BELMONTE, C.,
CERVERO, F. & HUNT, S.P. (1998). Altered nociception, analgesia
and aggression in the mice lacking the substance P receptor.
Nature, 392, 394 ± 397.
KURIHARA, T. & YOSHIOKA, K. (1996). The excitatory and
inhibitory modulation of primary a€erent ®bre-evoked responses
of ventral roots in the neonatal rat spinal cord exerted by nitric
oxide. Br. J. Pharmacol., 118, 1743 ± 1753.
MAZARIO, J. & HERRERO, J.F. (1999). Antinociceptive e€ects of
metamizol (dipyrone) in rat single motor units. Neurosci. Lett.,
273, 1 ± 14.
MAZARIO, J., GAITAN, G. & HERRERO, J.F. (2001). Cyclooxygenase-
1 vs cyclooxigenase-2 inhibitors in the induction of antinocicep-
tion in rodent withdrawal re¯exes. Neuropharmacology, 40, 937 ±
945.
NISHIHARA, I., MINAMI, T., WATANABE, Y., ITO, S. & HAYAISHI, O.
(1995). Prostaglandin E2 stimulates glutamate release from
synaptosomes of rat spinal cord. Neurosci. Lett., 196, 57 ± 60.
OCHS, H.R., GREENBLATT, D.J., ABERNETHY, D.R., ARENDT, R.M.,
GERLOFF, J., EICHELKRAUT, W. & HAHN, N. (1985). Cere-
brospinal ¯uid uptake and peripheral distribution of centrally
acting drugs: relation to lipid solubility. J. Pharm. Pharmacol.,
37, 428 ± 431.
DEL SOLDATO, P., SORRENTINO, R.
& PINTO, A. (1999). NO-
aspirins: a class of new anti-in¯ammatory and antithrombotic
agents. TiPS, 20, 319 ± 323.
DICKENSON, A.H. & SULLIVAN, A.F. (1987). Evidence for a role of
the NMDA receptor in the frequency dependent potentiation of
deep rat dorsal horn nociceptive neurones following C ®bre
stimulation. Neuropharmacology, 26, 1235 ± 1238.
FIORUCCI, S. (2001). NO-releasing NSAIDs are caspase inhibitors.
Trends in Immunology, 22, 232 ± 235.
FUTTER, L.E., AL-SWAYEH, O.A. & MOORE, P.K. (2001). A
comparison of the e€ect of nitroparacetamol and paracetamol
on liver injury. Br. J. Pharmacol., 132, 10 ± 12.
& HERRERO, J.F. (2002). Sube€ective doses of
GAITAN, G.
dexketoprofen trometamol enhance the potency and duration
of fentanyl antinociception. Br. J. Pharmacol., (in press).
HERRERO, J. F. & CERVERO, F. (1996a). Changes in nociceptive
re¯ex facilitation during carrageenan-induced arthritis. Brain
Res., 717, 62 ± 68.
PELISSIER, T., ALLOUI, A., PAEILE, C. & ESCHALIER, A. (1995).
Evidence for a central antinociceptive e€ect of paracetamol
involving spinal 5HT3 receptors. Neuroreport, 6, 1546 ± 1548.
PINI, L.A., VITALE, G., OTTANI, A.
& SANDRINI, M. (1997).
HERRERO, J. F. & CERVERO, F. (1996b). Supraspinal in¯uences on
the facilitation of rat nociceptive re¯exes induced by carrageenan
monoarthritis. Neurosci Lett., 209, 21 ± 24.
HERRERO, J.F. & HEADLEY, P.M. (1991). The e€ects of sham and
full spinalization on the systemic potency of m- and k-opioids on
spinal nociceptive re¯exes in rats. Br. J. Pharmacol., 104, 166 ±
170.
HERRERO, J.F. & HEADLEY, P.M. (1996). Reversal by naloxone of
the spinal antinociceptive actions of a systemically administered
NSAID. Br. J. Pharmacol., 118, 968 ± 972.
Naloxone-reversible antinociception by paracetamol in the rat.
J. Pharmacol. Exp. Ther., 280, 934 ± 940.
ROMERO-SANDOVAL, A., MAZARIO, J. & HERRERO, J.F. (2001).
Comparison of the analgesic e€ects of paracetamol and NO-
paracetamol in rat single motor units. In¯amm. Res., 50, S211.
SOLANO, R.E. & HERRERO, J.F. (1997). Cutaneous responsiveness of
rat single motor units activated by natural stimulation. J.
Neurosci. Methods, 73, 135 ± 140.
SOUSA, A.M. & PRADO, W.A. (2001). The dual e€ect of a nitric oxide
donor in nociception. Brain Res., 897, 9 ± 19.
HERRERO, J.F., LAIRD, J.M.A. & LOPEZ-GARCIA, J.A. (2000). Wind-
up of spinal cord neurones and pain sensation: much ado about
something? Prog. Neurobiol., 61, 169 ± 203.
HERRERO, J.F., THORN, S.A. & HEADLEY, P.M. (1996). Relative
peripheral, spinal and supraspinal actions of a non-steroidal anti-
in¯ammatory drug (¯unixin) compared to a mu-opioid agonist
(fentanyl) on nociceptive re¯exes in the rat. Analgesia, 2, 11 ± 18.
HRABIE, J.A., KLOSE, J.R., WINK, D.A. & KEEFER, L.K. (1993). New
nitric oxide-releasing zwitterions derived from polyamines. J.
Org. Chem., 58, 1472 ± 1476.
THORN, S.A., HERRERO, J.F. & HEADLEY, P.M. (1994). Stimulus
intensity and the comparative ecacy of m- and k-opioid agonists
on nociceptive spinal re¯exes in the rat. Brain Res., 663, 352 ±
356.
WALLACE, J.L., DEL SOLDATO, P., CIRINO, G. & MUSCARA, M.N.
(1999). Nitric oxide-releasing NSAIDs: GI-safe antithrombotics.
Idrugs, 2, 321 ± 326.
(Received December 11, 2001
Revised December 27, 2001
Accepted January 4, 2002)
British Journal of Pharmacology vol 135 (6)