ORIGINAL ARTICLE

Antinociceptive effect of systemically administered dipyrone

(metamizol), magnesium chloride or both in a murine model of
cancer
B.E. Brito1, E. Vazquez2,3, P. Taylor1, Y. Alvarado4, H. Vanegas2, A. Millan5, V. Tortorici2,3,5
1 Laboratory of Cellular and Molecular Pathology, Center for Experimental Medicine, Venezuelan Institute for Scientific Research (IVIC), Caracas,
Bolivarian Republic of Venezuela
2 Laboratory of Neurophysiology, Center for Biophysics and Biochemistry, Venezuelan Institute for Scientific Research (IVIC), Caracas, Bolivarian
Republic of Venezuela
3 School of Psychology, Andr
es Bello Catholic University, Caracas, Bolivarian Republic of Venezuela
4 Laboratory of Molecular Characterization and Biomolecules, Department of Research Materials, Technology and Environment, Venezuelan
Institute for Scientific Research (IVIC), Caracas, Bolivarian Republic of Venezuela
5 Department of Behavioral Sciences, Metropolitan University (UNIMET), Caracas, Bolivarian Republic of Venezuela

Correspondence Abstract
V
ıctor Tortorici
E-mail: victortortorici@gmail.com Background: Opioid effectiveness to treat cancer pain is often
compromised by the development of tolerance and the occurrence of
Funding sources undesirable side effects, particularly during long-term treatment. Hence,
This project was supported by the Mission the search for more efficient analgesics remains a necessity. The main
Science Program Grant Project 2007000881
goal of this study was to relieve neuropathic symptoms associated with
from the Ministry of Science, Technology
tumour growth by administering the non-opioid analgesic dipyrone
and Innovation and by a grant from the
company ENI DACION-BV, according to the (DIP) alone or in combination with magnesium chloride (MgCl2), an
Law on Science, Technology and Innovation adjuvant that blocks the NMDA receptor channel.
(LOCTI) of the Bolivarian Republic of Vene- Methods: Mice were inoculated with a melanoma cell line (B16-BL6)
zuela. in the left thigh and two protocols were used to evaluate the effect of
DIP (270 mg/kg), MgCl2 (200 mg/kg), or the combination DIP-MgCl2. In
Conflicts of interest
the therapeutic protocol the drugs, alone or combined, were
None declared.
administered once tumour had promoted increased nociception. In the
preventive protocol, drugs were administered prior to the appearance of
Accepted for publication the primary tumour. Tumour growth was assessed with a caliper and
8 August 2016 nociception was determined using behavioural tests.
Results: DIP promoted antinociception only at the beginning of both
doi:10.1002/ejp.957 protocols due to the development of tolerance. The combination DIP-
MgCl2 improved the antinociceptive effect, avoiding tolerance and
reducing tumour growth in the preventive treatment, more efficiently
than each compound alone.
Conclusions: These results suggest that DIP-MgCl2 may represent a
safe, affordable and accessible option to reduce tumour growth and to
treat cancer pain avoiding the risk of tolerance, without the typical
complications of opioids agents, particularly when long-term treatment
is required.
Significance: This study shows a non-opioid analgesic combined with
an adjuvant as a therapeutic option to treat cancer pain. The avoidance
of antinociceptive tolerance when repeated administration is required, as
well as tumor growth reduction, are additional advantages to be
considered.

© 2016 European Pain Federation - EFICâ Eur J Pain  (2016) – 1

Dipyrone combined with MgCl2 for cancer pain B.E. Brito et al.

Here, we investigated whether repeated DIP

1. Introduction
administration reduces neuropathy manifestations
Cancer pain usually implies a prolonged use of anal- induced by the growth of a tumour engulfing the
gesics and complications associated to their repeated sciatic nerve. The combination MgCl2-DIP reversed
administration (Foley, 2011; Portenoy, 2011; Mori the progressive decrease in pain threshold associated
et al., 2012). According to the WHO analgesic ladder with nerve compression and prevented tolerance. In
(WHO (World Health Organization), 1987), dipyrone the preventive protocol, both DIP and MgCl2 signifi-
(DIP, metamizol), a non-opioid analgesic and anti- cantly reduced the tumour mass, but this reduction
pyretic drug used in Latin America and Europe was greater with MgCl2-DIP coadministration.
(IASP, 1992), can be used in the early oncological
stages. DIP prevents nociceptor sensitization by
inhibiting cyclooxygenases-1-3 (Campos et al., 2. Methods
1999). DIP also inhibits cyclooxygenase activity in
the neuraxis (Bannwarth et al., 1995) and blocks 2.1 Animals
the ascending nociceptive message (Jurna and Helm-
In all experiments, male adult C57BL/6 mice of 20–
reich, 1986). DIP antinociceptive action also involves
25 g (n = 10/group) were used. The animals were
the inhibition of glutamate release and glutamate
bred at the Central Animal Facility of the Venezue-
binding to N-methyl-D-aspartate (NMDA) receptors
lan Institute for Scientific Research (IVIC), and were
(Beirith et al., 1998; Siebel et al., 2004). Its
handled according to the Bioethical Guidelines of
microinjection into the mesencephalic periaqueduc-
the Society for Neuroscience. The experimental pro-
tal gray (PAG), or the rostroventromedial medulla
tocol was approved by the Bioethical Committee for
(RVM), key stations of the descending pain modula-
the Use of Experimental Animals (COBIANIM) of
tory system, produces antinociception (Carlsson
IVIC. The animals were kept in groups of five in
et al., 1986; Tortorici and Vanegas, 1994, 2000;
plastic boxes with a microisolator filter on top. The
Vazquez et al., 2005; Vanegas and Tortorici, 2007;
colony room temperature was kept constant at 22 °C
Tortorici et al., 2009) which is apparently mediated
and 25 air changes were adjusted per hour. The
by endogenous opioids as it may be reversed either
humidity was also regulated and light/dark cycles
by naloxone (Vlaskovska and Krushkov, 1989; Tor-
lasted 12 h, respectively. All experiments were per-
torici et al., 1996; Vanegas et al., 1997; Vanegas
formed during the light cycle. Bedding from each
and Tortorici, 2002; Hern
andez-Delgadillo and Cruz,
cage and food (Ratarina, Alimentos Protinal, C.A.,
2006), or by CTOP (Tortorici et al., 2009). As with
Valencia, Venezuela) were sterilized in a Cobalt-60
opioids, repeated administration of DIP induces
Plant at IVIC. The water was subjected to a double
tolerance to its antinociceptive effect (Tortorici and
filtration process and like the food, was available
Vanegas, 2000; Vanegas and Tortorici, 2002;
ad libitum. All assays were performed in the Labora-
Tortorici et al., 2004, 2009; Yilmaz and Ulugol,
tory of Neurophysiology, of the Center for Bio-
2009) apparently involving endogenous antiopioid
physics and Biochemistry (CBB) at IVIC, under the
agents (Tortorici et al., 2004; Hern
andez-Delgadillo
supervision of a responsible experimenter with the
and Cruz, 2006) and possibly an opioid-like activa-
advice of the veterinary staff of IVIC. At the end of
tion of NMDA receptors (Trujillo and Akil, 1991;
each experiment the animals were euthanized with
Price et al., 2000).
an overdose of sodium thiopental (SM Pharma Labo-
Adjuvants such as MgCl2 have also promoted
ratories, Caracas, Venezuela), following the recom-
antinociception in neuropathic pain models and
mendations of the Panel on Euthanasia of the
analgesia in the clinical practice. In rats with chronic
American Veterinary Medical Association (AVMA,
constriction injury of the sciatic nerve, administra-
2013).
tion of either MgCl2-morphine or MgCl2-DIP pre-
vented the development of tolerance after repeated
2.2 Drugs used
administration and potentiated their antinociceptive
effects (Begon et al., 2002; Ulugol et al., 2002; Dipyrone (DIP), (methylamino-antipyrine-4-sodium
G
omez-Barrios et al., 2006; G
omez-Barrios and methanesulphonate), also known as metamizol, was
Tortorici, 2007). Mg2+ promotes postoperative pain kindly donated by Laboratorios Biotech, C.A., Cara-
relief in humans (Kara et al., 2002; Albrecht et al., cas, Venezuela. A dose of 270 mg/kg was injected
2013), reducing opioid rescue doses during surgery intraperitoneally (i.p.). This was the lowest effective
or in recovery. dose when used in our pilot experiments with

2 Eur J Pain  (2016) – © 2016 European Pain Federation - EFICâ

B.E. Brito et al.
C57BL/6 mice. The drug was in its pure state which
avoided additional experiments to monitor the
potential effects of excipients. Its maximum effect
(at 30 min post-injection) was determined in previ-
ous acute experiments of 120 min duration (data
not shown here for space reasons). In those experi-
ments the behavioural tests (see below) were
applied with an interval of 15 min between time-
points. Magnesiun chloride (MgCl2) was used in its
hexahydrated presentation, with 95% purity
(MgCl2
6H2O, Sigma-Aldrich Laboratories, USA). A
dose of 200 mg/kg was i.p. administered. This dose
was chosen based on other groups’ work (Woolf and
Thompson, 1991; Begon et al., 2002) and also based
on our previous experience (G
omez-Barrios et al.,
2006; G
omez-Barrios and Tortorici, 2007). Its maxi-
mum effect (at 60 min after being injected) was
recorded using the same approach as with DIP alone
(see above).
When combining both agents, MgCl2 was initially
injected and 30 min later DIP was administered to
ensure the coincidence of both maximum effects.
Drugs were dissolved in physiological solution (0.9%
NaCl; Laboratorios Behrens, Caracas, Venezuela) and
0.2 mL was the injection volume. An equivalent
volume of saline was administered i.p. to control
animals.
2.3 Experimental model
Our experimental model was designed to quantify
the nociceptive behavioural consequences of a
tumour–nerve interaction and its possible pharmaco-
logical management. It involved the use of cells of
the highly invasive B16–BL6 melanoma line
employed by Sasamura et al. (2002) in their skin
cancer pain model in C57BL/6 mice.
After light sedation, obtained by placing each ani-
mal in a ventilatory capsule containing halothane
gas (Hoechst Marion Roussel, S.A., Caracas, Vene-
zuela), C57BL/6 mice were inoculated intramuscu-
larly (i.m.) in the left mid-thigh (105 cells/100 lL/
animal), in the immediate proximity of the sciatic
nerve as in the neuropathic cancer pain model pro-
posed by Shimoyama et al. (2002).
The cell line was cultured, using conventional
techniques, in Dulbecco’s modified Eagle’s medium-
low glucose (Sigma-Aldrich Laboratories) with 5%
foetal bovine serum. The tumour grew predictably
with time and gradually compressed the nerve, caus-
ing nerve injury that resembled a neurotropic mela-
noma or a melanotic schwannoma growing around
the sciatic nerve.
© 2016 European Pain Federation - EFICâ
Dipyrone combined with MgCl2 for cancer pain
2.4 Determination of tumour growth
Because the tumour in the thigh tended to grow
acquiring an elliptical shape, its volume was deter-
mined using a calibrated caliper and the ellipse vol-
ume formula: V = 4/3p (rm 9 rs2). In this formula V
corresponds to the tumour volume expressed in cm3,
rm is the largest radius of the two measurements
while rs is the smaller. These measurements were
performed in lightly sedated animals (halothane
vapours, see above) to minimize the stress associated
with handling.
2.5 Behavioural tests
The von Frey and Hargreaves tests were applied to
the hind paws and withdrawal responses were
observed before (baseline, BL) and after inoculation
of the cell line, as well as before and after drug
administration or their respective control vehicle.
The von Frey test involves the application of a series
of nylon monofilaments (Touch-Test Sensory Evalua-
tor, Semmes-Weinstein monofilaments, Stoelting, IL,
USA), to the plantar region of the paw in order to
apply a specific bending pressure depending on the
calibrated filament used. This pressure was expressed
in grams. The filaments were applied in ascending or
descending sequence to determine the minimum
pressure that evoked a clear withdrawal response.
This procedure was performed three times, with
3 min between tests so that the results expressed in
the graphs are an average of these measurements. The
response of the contralateral limb (not inoculated)
served as internal control.
The Hargreaves test involves placing each animal in
a paw analgesiometer (Hargreaves, IITC Life Science,
Series 8, CA, USA). The equipment has a transparent
plastic chamber with a glass floor preheated to 28–
30 °C. The animals remain in the chamber for a
habituation period of 5 min. Then, a thermal noci-
ceptive stimulus was applied using an incandescent
light source with an illumination area of 7 mm2. The
time it took the animal to withdraw its paw from the
radiant heat beam (latency) was determined. Cut-off
time was 30 s. This procedure was applied three times
with a 5 min separation between trials so that the
results shown in the graphs represent an average of
the three measures. The unaffected contralateral paw
served as internal control.
2.6 Drug administration protocol
To evaluate the single or combined effect of the
drugs, each group of mice received from a blinded
Eur J Pain  (2016) – 3
Dipyrone combined with MgCl2 for cancer pain B.E. Brito et al.

experimenter a single i.p. injection of the agents, Median tests were performed to verify data normal-
every other day for 4 days, to minimize the effect of ity. Thereafter, differences between groups were
excessive handling. For the therapeutic treatment, evaluated by applying the Student’s t-test or the
drugs were administered on days 11, 13, 15 and 17 one-way analysis of variance (ANOVA). Where
post-inoculation. This period corresponded to the appropriate, Bonferroni’s multiple comparison was
maximum expression of tumour growth and to the used to evaluate differences between specific treat-
major changes in pain threshold. The preventive ments. Differences were acknowledged as statistically
treatment was carried out on days 1, 3, 5 and 7 significant when p ≤ 0.05. The statistical program
post-inoculation, before the primary tumour could used was GraphPad Prism version 5.0 (GraphPad
be detected and before the appearance of changes in Software, CA, USA).
pain threshold. This latter protocol was intended to
assess the possible existence of a ‘critical time-win-
dow’, as a pharmacological way to intervene before 3. Results
the onset of pain symptoms.
3.1 Changes in the cell-inoculated leg
2.7 Statistical analysis
As shown in Fig. 1(a, b), BL for the von Frey test
The results were reported as the mean  SE of the was 3.48  0.36 g and for the Hargreaves test
sample. The Kolmogorov–Smirnov and Levene 6.51  0.54 s. Nociceptive responsiveness increased

Dipyrone + magnesium Dipyrone

Magnesium Saline
Treatment Treatment
(a) days (i.p.)
(c)
days (i.p.)
Paw withdrawal threshold (g)

Paw withdrawal threshold (g)

8 8
von Frey's test

6
von Frey's test

6
** ** ** **
4 4

2 2

0 ** 0
BL 1 3 5 7 9 11 13 15 17 Days BL 1 3 5 7 9 11 13 15 17 Days
Cell line (i.m.) Cell line (i.m.)

Treatment Treatment
(b) days (i.p.)
(d) days (i.p.)
16 16
Paw withdrawal latency (s)

Paw withdrawal latency (s)

Hargreave's test

Hargreave's test

12
** ** 12 ** **
8 8

4 4
**
0 0
BL 1 3 5 7 9 11 13 15 17 Days BL 1 3 5 7 9 11 13 15 17 Days

Cell line (i.m.) Cell line (i.m.)

Figure 1 Effect of therapeutic administration (days 11–17, grey box) of dipyrone (270 mg/kg), magnesium (200 mg/kg), or both, on changes in
nociceptive responsiveness caused by tumour progression in the cell-inoculated leg (a, b), or the contralateral, not inoculated, leg (c, d). Above:
von Frey tests. Below: Hargreaves tests. BL: baseline (response value before inoculation of the cell line), CL: cell line, i.m.: intramuscular injection,
i.p.: intraperitoneal injection. **p ≤ 0.01, n = 10 mice/group.

4 Eur J Pain  (2016) – © 2016 European Pain Federation - EFICâ

B.E. Brito et al.
gradually from day 3 after i.m. inoculation of the
melanoma cell line, reaching 1.78  0.38 g on day 9
for the withdrawal threshold to mechanical stimula-
tion of the paw and 3.80  0.36 s of withdrawal
latency in the affected limb to thermal stimulation.
These changes (p ≤ 0.01 for both tests, day 9 vs. BL)
coincide with the moment when the presence of the
primary tumour can be noticed by touching the
limb. From that moment on, the response tended to
stabilize, reaching at the end of the experiment
1.01  0.42 g and 2.34  0.70 s, respectively, in the
group receiving the vehicle.
3.2 Effect of therapeutic administration of
dipyrone, magnesium or both upon the
changes produced by tumour progression
On day 11, treatment with MgCl2 returned the
responses to magnitudes equivalent to BL levels
(3.29  0.43 g and 6.79  0.57 s, respectively;
p ≥ 0.05 for both tests, day 11, MgCl2 vs. baseline).
However, this effect decreased with successive admin-
istrations (Fig. 1a, b), to the point that on day 15
responses for the von Frey and Hargreaves tests were
1.34  0.22 g and 3.23  0.28 s, respectively. Fol-
lowing that day the responses were almost identical to
those of the saline group (p ≥ 0.05 for both tests, days
15 and 17, MgCl2 vs. saline). DIP treatment also
caused a significant decrease in nociception in both
tests at the start of treatment (5.22  0.38 g and
10.78  0.90 s, respectively; p ≤ 0.01 for both tests
vs. BL). This antinociceptive effect gradually
decreased (Fig. 1a, b), such that on day 17 the
response values were similar to those obtained in the
saline group (1.62  0.45 g and 4.00  0.42 s;
p ≥ 0.05 for both tests, day 17, DIP vs. saline). The
group receiving the combination MgCl2-DIP also
obtained a marked antinociceptive effect when initiat-
ing the protocol (5.93  0.31 g and 12.26  0.70 s;
p ≤ 0.01 for both tests, day 11, MgCl2-DIP vs. BL),
which was slightly higher than that produced initially
by DIP. However, in this case the effect was main-
tained throughout the treatment (Fig. 1a, b), so that
the combined administration of both agents not only
produced the best antinociceptive effect but also pre-
vented the decrease in antinociception.
The increase in nociceptive responsiveness in the
inoculated limbs was not observed in the contralat-
eral legs. As shown in Fig. 1(c, d), the responses
remained constant until the end of treatment, except
for the groups receiving DIP or the combination
MgCl2-DIP. In these cases, the treatment produced
on day 11 a marked antinociceptive effect, i.e.,
© 2016 European Pain Federation - EFICâ
Dipyrone combined with MgCl2 for cancer pain
5.80  0.36 g and 12.12  0.66 s (p ≤ 0.01 for both
tests; day 11, DIP and MgCl2-DIP vs. BL). Again, this
antinociceptive effect progressively decayed in the
group receiving only DIP and on day 17 the
responses to both tests showed values similar to
those detected in the saline control (4.00  0.53 g
and 7.71  0.81 s; p ≥ 0.05 for both tests, day 17,
DIP vs. saline). By contrast, the antinociceptive effect
caused by the combination MgCl2-DIP was main-
tained throughout the treatment.
3.3 Effect of preventive administration of
dipyrone, magnesium or both upon the
changes produced by tumour progression
Fig. 2(a, b) shows a progressive nociceptive increase
after post-inoculation day 3 with both tests for the
saline control, which tended to stabilize from day 11
until the end of treatment (1.37  0.21 g and
3.70  0.55 s, respectively; p ≤ 0.01 for both tests,
days 11 and 17, saline vs. BL). This nociceptive
increase suffered a delay until day 11 in the group
receiving MgCl2 (p ≥ 0.05 for both tests, day 11,
MgCl2 vs. BL) which represents a 144-h deferral in
the onset of ‘mechanical allodynia’ and ‘thermal
hyperalgesia’. However, this effect decreased follow-
ing that day, and on day 17 reached values similar
to those of the saline group (1.82  0.27 g and
2.99  0.30 s; p ≥ 0.05, day 17, MgCl2 vs. saline).
The group receiving the preventive treatment with
DIP showed a significant antinociceptive effect only
at the beginning of the protocol (4.78  0.32 g and
10.51  0.63 s; p ≤ 0.01, day 1, BL vs. DIP). How-
ever, this effect diminished progressively to reach
values equivalent to BL at the end of treatment
(2.72  0.37 g and 7.29  0.60 s values; p ≥ 0.05,
day 7, DIP vs. BL) and continued to fall tending to
be similar to the saline group at the end of the
experiment 1.60  0.26 g and 2.83  0.31 s;
p ≥ 0.05, day 17, DIP vs. saline). The preventive
treatment with the combination MgCl2 -DIP also
produced a marked antinociceptive effect, which was
even greater than that shown by a single administra-
tion of DIP (5.75  0.32 g and 13.06  0.66 s;
p ≤ 0.05, day 1, MgCl2-DIP vs. DIP and p ≤ 0.001,
MgCl2-DIP vs. BL). This effect was maintained
throughout the treatment protocol. Once the admin-
istration of MgCl2-DIP was discontinued a gradual
increase in nociception was observed to reach on
day 17 values similar to BL (3.15  0.26 g and
6.33  0.47 s; p ≥ 0.05 for both tests, day 17,
MgCl2-DIP vs. BL). These results indicate that early
administration of the combination MgCl2-DIP
Eur J Pain  (2016) – 5
Dipyrone combined with MgCl2 for cancer pain B.E. Brito et al.

Dipyrone + magnesium Dipyrone

Magnesium Saline

(a) Treatment (c) Treatment

days (i.p.) days (i.p.)
Paw withdrawal threshold (g)

Paw withdrawal threshold (g)

8 8
von Frey's test

von Frey's test

6 6
*** *** ***
** ** **
**
4 ns 4

2 2
*
0 ** 0
BL 1 3 5 7 9 11 13 15 17 Days BL 1 3 5 7 9 11 13 15 17 Days

Cell line (i.m.) Cell line (i.m.)

(b) Treatment (d) Treatment

days (i.p.) days (i.p.)
16 16

Paw withdrawal latency (s)

Paw withdrawal latency (s)

*** ***
Hargreave's test
Hargreave's test

12 *** 12
** ** **
ns
8 8

4 4
*
**
0 0
BL 1 3 5 7 9 11 13 15 17 Days BL 1 3 5 7 9 11 13 15 17 Days

Cell line (i.m.) Cell line (i.m.)

Figure 2 Effect of preventive administration (days 1–7, grey box) of dipyrone (270 mg/kg), magnesium (200 mg/kg), or both, on changes in noci-
ceptive responsiveness caused by tumour progression in the cell-inoculated leg (a, b), or the contralateral, not inoculated, leg (c, d). Above: von
Frey tests. Below: Hargreaves tests. BL: baseline (response value before inoculation of the cell line), CL: cell line, i.m.: intramuscular injection, i.p.:
intraperitoneal injection, ns: no significant. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, n = 10 mice/group.

produced antinociception and prevented the onset of values obtained by DIP (5.67  0.26 g and
‘mechanical allodynia’ and ‘thermal hyperalgesia’ 12.93  0.76 s; p ≤ 0.05 for both tests, day 1,
throughout treatment. MgCl2-DIP vs. DIP), but also avoided the develop-
On Fig. 2(c, d) it can be seen, again, that the ment of tolerance. When treatment was stopped, a
effects of the melanoma cell line were only observed progressive decay of the effect was observed, reach-
in the inoculated limb. Only the administration of ing values similar to BL at the end of the experiment
DIP or the combination MgCl2-DIP achieved a (3.90  0.33 g and 8.51  0.71 s, p ≥ 0.05, day 17,
detectable level of antinociception in the unaffected MgCl2-DIP vs. BL).
limb. On day 1, the effect of DIP produced values
significantly higher than BL (5.00  0.53 g and
3.4 Effect of different treatments on tumour
10.22  0.90 s; p ≤ 0.01 for both tests, day 1, DIP
growth
vs. BL), but this antinociceptive effect gradually
declined during the days of administration, so that As shown on Fig. 3(a), the primary tumour was
by day 7 the results were similar to BL figures detectable from day 9 post-inoculation. It can also be
(3.30  0.37 g and 7.88  0.67 s; p ≥ 0.05 for both seen that the different treatments used in the thera-
tests, day 7, DIP vs. BL). Meanwhile, the combined peutic protocol did not induce changes in the pattern
administration of MgCl2-DIP not only caused a of tumour growth when compared with the saline
marked antinociception, which again overcame the control group (p ≥ 0.05 for all three treatments vs.

6 Eur J Pain  (2016) – © 2016 European Pain Federation - EFICâ

B.E. Brito et al. Dipyrone combined with MgCl2 for cancer pain

Dipyrone + magnesium and that observed in the group receiving saline

Magnesium (p ≤ 0.05, day 17, both single treatments vs. saline
Treatment
(a) Dipyrone days (i.p.) and vs. MgCl2-DIP).
Saline
4
4. Discussion and conclusions
Tumor volume (cm3)

3
Pain, when chronic, defeats its biological function and
leads to impairments in the quality of life. About 95%
2 of patients with advanced cancer suffer from this con-
Primary
tumor dition (Portenoy, 1989) and, despite countless efforts,
1 its continued relief still remains a public health prob-
lem (Mori et al., 2012). The selection of an analgesic
0 critically depends on the intensity of pain (WHO,
BL 1 3 5 7 9 11 13 15 17 Days 1987). However, sooner or later opioids become the
Cell line (i.m.)
reference choice to relieve cancer pain (Bitros, 2005;
Fallon et al., 2006), despite their limited access and
Treatment side effects of prolonged administration. Thus, the
(b) days (i.p.) search for safe, more efficient and affordable analgesics
4 remains a necessity. One way to contribute to this
** search is the use non-opioid analgesics, alone or with
Tumor volume (cm3)

3 adjuvants, instead of using weak or strong opioids

2 Primary
tumor
1
0 tries of the European Union and Latin America, it is
BL 1 3 5 7 9 11 13
Cell line (i.m.)
Figure 3 Effect of dipyrone (270 mg/kg), magnesium (200 mg/kg), or
both, on tumour growth. (a) Therapeutic protocol (days 11–17, grey
box). (b) Preventive protocol (days 1–7, grey box). BL: baseline (re-
sponse value before inoculation of the cell line), CL: cell line, i.m.:
intramuscular injection, i.p.: intraperitoneal injection. *p ≤ 0.05,
**p ≤ 0.01, n = 10 mice/group.
saline). Conversely, when the preventive protocol
was administered (Fig. 3b), differences in tumour
progression were observed. The group that received
the combination MgCl2-DIP was the one with the
lowest mass growth at the end of the experiment
(0.96  0.29 cm3) compared with the saline group
(3.69  0.25 cm3). This significant reduction in
tumour growth (p ≤ 0.01, day 17, MgCl2 -DIP vs.
saline) showed that, in addition to causing an
improved level of antinociception and the avoidance
of tolerance, the early administration of both agents
also promoted the lowest level of tumour expansion.
The single administration of DIP or MgCl2 also led to
a lower level of tumour growth (1.88  0.31 and
2.02  0.31 cm3, respectively), which stood at an
intermediate level between the effect of MgCl2-DIP
throughout the disease. The present study thus sought
to relieve, or even prevent, neuropathic nociception
* and tumour growth by administering either DIP, an
adjuvant such as MgCl2, or a combination of both.
DIP is commonly used to treat postoperative pain,
colic pain, migraine and cancer pain. In many coun-
15 17 Days
a popular non-opioid, non-NSAID, first-line anal-
gesic. In some countries, however, DIP has been
banned because of the alleged risk of agranulocytosis
(see K€ otter et al., 2015; for review). However, the
incidence of DIP-induced agranulocytosis is extre-
mely rare (one case/million/treatment) (Kaufman
et al., 1996; Ibanez et al., 2005). Some genetic, or
even epigenetic, predisposition could not be
excluded, since DIP appears to be associated with
agranulocytosis in certain regions of the world but
much less so in others (Nikolova et al., 2013). A
recent systematic review and meta-analysis including
79 trials and almost 4000 patients, reported only few
serious adverse events, with no difference between
DIP and other analgesics such as opioids, paraceta-
mol and NSAIDs. More importantly, no deaths or
agranulocytosis were reported (K€ otter et al., 2015).
According to our previous dose–response curves in
rats, doses as high as 200 or 400 mg/kg of DIP are
required to produce antinociception (Tortorici and
Vanegas, 1994). In agreement with the equation of
Pong et al. (1985), an oral dose of 200 mg/kg of DIP
in mice would be equivalent to a 500 mg oral dose
for adult humans, which is similar or even lower to
that recommended by the manufacturer [Bral

© 2016 European Pain Federation - EFICâ Eur J Pain  (2016) – 7

Dipyrone combined with MgCl2 for cancer pain
(Laboratorios Biotech, C.A.), 0.5–1.0 g, see Lehr
et al., 2007/2008] or the IASP (1992) to treat acute
pain. If these relationships can be taken as accept-
able indicators, the dose used in our study can be
deemed as equivalent to a human therapeutic dose.
Additionally, when used to treat cancer pain, DIP at
either 1 or 2 g doses every 8 h tended to be better
tolerated than 10 mg of morphine every 4 h, indi-
cating that the use of high doses of orally adminis-
tered DIP to relieve severe pain, including chronic
cancer pain, is a common practice in several coun-
tries and is clinically safe compared to other com-
monly used analgesics (Rodr
ıguez et al., 1994).
DIP shares properties attributable to opioid anal-
gesics. Its action can be antagonized by naloxone, an
opioid antagonist (Tortorici et al., 1996), or by
CTOP, a specific l-opioid receptor antagonist (Tor-
torici et al., 2009). In addition, its repeated adminis-
tration leads to a loss of analgesic efficacy due to the
development of tolerance (Tortorici and Vanegas,
2000; Vanegas and Tortorici, 2007; Tortorici et al.,
2009) which apparently involves endogenous antio-
pioid agents such as cholecystokinin (Tortorici et al.,
2004) and probably an opioid-like (Trujillo and Akil,
1991; Price et al., 2000) activation of NMDA recep-
tors. Here, we used DIP to relieve hyperalgesia asso-
ciated with the expansion of a tumour engulfing the
sciatic nerve. MgCl2 was used to prevent tolerance
during repeated administration. This agent blocks
the NMDA receptor channel, preventing cation
influx and the consequent pro-tolerance intracellular
cascades (Bryant et al., 2006; Mendez and Trujillo,
2008). DIP-MgCl2 not only produced the best
antinociceptive effect but also avoided tolerance.
This effect was observed when applying the treat-
ment therapeutically, or after applying it preven-
tively. The fact that a similar behavioural response
can be evoked in the ‘healthy’ contralateral paws
used herein as internal controls would argue in
favour of a lack of drug action (tolerance) instead of
hyperalgesia as the cause of effect decrease.
Preventive analgesia involves the administration in
advance of an analgesic therapy before the stimulus
that generates pain occurs (McQuay, 1992; Katz and
McCartney, 2002). Here, the appearance of the pri-
mary tumour was the time-reference event (around
day 9). Hence, it was decided to schedule the preven-
tive administration on days 1, 3, 5 and 7. Preventive
MgCl2-DIP administration not only produced the best
antinociceptive effect but also prevented ‘mechanical
allodynia’ and ‘thermal hyperalgesia’ after discontin-
uing the treatment, conferring an extended
protection against ‘pain’ by nerve compression.
8 Eur J Pain  (2016) –
B.E. Brito et al.
Importantly, MgCl2 administered individually only
produced an antihyperalgesic effect when adminis-
tered preventively, avoiding changes in pain thresh-
old until day 11 post-inoculation. Also significant was
the reduction in tumour growth by the preventive
treatment. MgCl2-DIP again yielded the best effect,
although both agents separately also presented com-
parative advantages.
Recent evidence suggests that glutamate plays an
important role in carcinogenesis. Glutamate activates
metabotropic and ionotropic receptors, both of which
have been implicated in signalling pathways in can-
cer (Willard and Koochekpour, 2013). In fact, inhibi-
tion of glutamate release or glutamate receptor
activation has proven to decrease growth, migration
and invasion, and to induce apoptosis in different
types of cancer (Willard and Koochekpour, 2013).
More specifically, the metabotropic glutamate type 1
(mGlu1) has been studied in cellular transformation
protocols employing melanoma models, showing
that an aberrant (ectopic) expression of mGlu1 is
sufficient to induce spontaneous melanoma develop-
ment (Shin et al., 2008). Also, the blockade of
mGlu1 and the use of NMDA receptor antagonists
inhibited melanoma growth (Song et al., 2012). In
the present study, a similar blockade might be occur-
ring preventively after using DIP, MgCl2, or their
combination. This still needs to be proven but can be
a possible explanation for the concomitant tumour
growth control and reversal of ‘allodynia and hyper-
algesia’. In addition, glutamate, acting through the
NMDA receptors, has been implicated in changes of
the natural killer cell response (Kuo et al., 2001),
which, through their cytotoxic activity, are endoge-
nously responsible for reducing tumour activity.
If other NMDA acting compounds, such as keta-
mine, may have similar findings remains to be eluci-
dated. Also, the effect of blocking NMDA channels
over sustained administrations deserves to be investi-
gated. On this regard, DIP may represent again a
safer way to modulate the NMDA activity. Several
reports remarked the absence of neurological adverse
events associated with DIP administration (such as
those induced by ketamine), including comparative
studies using morphine as the reference (K€ otter
et al., 2015). These observations also imply that DIP
can be used after discharge, without the necessary
follow-up that ketamine requires during intra-hospi-
tal administration. On the other hand, the analgesic
efficacy of ketamine may depend on the types of
cancer and pain (see Andoh et al., 2008).
Our experimental model might resemble a clinical
complication due to compression or infiltration of
© 2016 European Pain Federation - EFICâ
B.E. Brito et al.
peripheral nerves by malignant tumours. Even
though metastases to individual nerves are considered
rare, it is possible to find melanoma neurotropism
causing infiltration or compression of nerve roots or
individual nerves, hence promoting tumour-related
compression neuropathy (Mandybur, 1974; Croker
et al., 2012). An alternative to the melanoma neu-
rotropism has been also proposed. Tumours arising
from neural crest-derived cells represent a heteroge-
neous group of neoplasms, including benign and
malignant tumours with melanocytic and schwannian
differentiation. Melanocytes and Schwann cells can
convert into each other, generating an intermediate
cell type, which expresses markers specific for mela-
nocytic and schwannian lineages (Dundr et al., 2009).
In fact, melanotic schwannomas or malignant melan-
otic schwannian tumours represent a rare type of
tumour that may involve spinal nerve roots (Torres-
Mora et al., 2014). These findings raise the possibility
that a similar type of nerve engulfing might occur
spontaneously. The genesis of such oncological situa-
tion does not necessarily need to come from a primary
distant location but, in turn, it may be associated to a
focal abnormal glial conversion (Yamate et al., 2003;
Cicuendez et al., 2012).
In summary, these results show that MgCl2-DIP
promoted a significant level of antinociception in
experimental cancer-induced pain. Hence, this combi-
nation may be an option to the use of opioids, with
the advantage of avoiding analgesic tolerance. Based
on the multiple mechanisms of action involved, the
use of DIP, alone or combined with MgCl2, can be
considered a multimodal approach for the treatment
of cancer pain. These findings may contribute to
increase the list of drugs with two or more different
mechanisms that have proven effective for the treat-
ment of pain while eliminating opioid unwanted side
effects [see Largent-Milnes et al. (2013), as a recent
example of innovative multifunctional compounds
efficacy]. According to our preventive treatment
results, cancer pain can and should be treated in
advance to ensure maximum control of the symptoms
and of tumour growth. Of course, early treatment
must match early detection through appropriately
implemented screening programs.
Acknowledgements
The authors want to thank the technical help of Alba
Becerra-Guevara during the acquisition of data. Also, the
assistance of the veterinary staff of IVIC’s Central Animal
Facility is greatly appreciated.
© 2016 European Pain Federation - EFICâ
Dipyrone combined with MgCl2 for cancer pain
Author contributions
Beatriz E. Brito: conception and design, culture techniques,
data acquisition, analysis, interpretation of data and draft-
ing the article. Enrique V
azquez and Anthony Mill
an:
interpretation of data and drafting the article. Peter Taylor,
Horacio Vanegas and Ysa
ıas Alvarado: critical revision for
important intellectual content. V
ıctor Tortorici: conception
and design, data acquisition, analysis, interpretation of data
and drafting the article; Corresponding author. All authors
discussed the results and commented on the manuscript.
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