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Dipyrone is locally hydrolyzed to 4-methylaminoantipyrine and its antihyperalgesic effect depends on CB2 and kappa-opioid receptors activation
Journal Pre-proof Dipyrone is locally hydrolyzed to 4-methylaminoantipyrine and its antihyperalgesic effect depends on CB2 and kappa-opioid receptors activation Gilson Gonçalves dos Santos, Willians Fernando Vieira, Pedro Henrique Vendramini, Bianca Bassani da Silva, Silviane Fernandes Magalhães, Cláudia Herrera Tambeli, Carlos Amilcar Parada PII:
S0014-2999(20)30097-2
DOI:
https://doi.org/10.1016/j.ejphar.2020.173005
Reference:
EJP 173005
To appear in:
European Journal of Pharmacology
Received Date: 26 November 2019 Revised Date:
3 February 2020
Accepted Date: 7 February 2020
Please cite this article as: Gonçalves dos Santos, G., Vieira, W.F., Vendramini, P.H., Bassani da Silva, B., Magalhães, S.F., Tambeli, Clá.Herrera., Parada, C.A., Dipyrone is locally hydrolyzed to 4methylaminoantipyrine and its antihyperalgesic effect depends on CB2 and kappa-opioid receptors activation, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/j.ejphar.2020.173005. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Dipyrone is locally hydrolyzed to 4-methylaminoantipyrine and its antihyperalgesic effect depends on CB2 and kappa-opioid receptors activation.
Gilson Gonçalves dos Santos¹*; Willians Fernando Vieira¹; Pedro Henrique Vendramini2; Bianca Bassani da Silva¹, Silviane Fernandes Magalhães¹; Cláudia Herrera Tambeli¹; Carlos Amilcar Parada¹
¹Department of Structural and Functional Biology, Institute of Biology, University of Campinas (UNICAMP), ZIP Code 13083-864, Campinas, Sao Paulo, Brazil; 2
ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of
Campinas (UNICAMP), ZIP Code 13083-970, Campinas, Sao Paulo, Brazil.
*Corresponding author: [email protected]. Full postal address: Department of Anesthesiology, University of California, San Diego, 9500 Gilman Drive, Mail Code 0818, La Jolla, CA 92093-0818
1 2 3
Dipyrone is locally hydrolyzed to 4-methylaminoantipyrine and its antihyperalgesic effect depends on CB2 and kappa-opioid receptors activation.
4 5
Gilson Gonçalves dos Santos¹*; Willians Fernando Vieira¹;
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Pedro Henrique Vendramini2; Bianca Bassani da Silva¹, Silviane Fernandes
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Magalhães; Cláudia Herrera Tambeli¹; Carlos Amilcar Parada¹
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¹Department of Structural and Functional Biology, Institute of Biology, University of
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Campinas (UNICAMP), ZIP Code 13083-864, Campinas, Sao Paulo, Brazil;
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2
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Campinas (UNICAMP), ZIP Code 13083-970, Campinas, Sao Paulo, Brazil.
ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of
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*Corresponding author: [email protected]. Full postal
14
address: Department of Anesthesiology, University of California, San Diego, 9500
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Gilman Drive, Mail Code 0818, La Jolla, CA 92093-0818
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Co-author’s e-mail addresses:
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W.F.V.: [email protected]
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P.H.V.: [email protected]
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B.B.S.: [email protected]
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S.F.M.: [email protected]
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C.H.T.: [email protected] C.A.P.: [email protected]
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Acknowledgements
26 27
1
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Abstract
29
Dipyrone is an analgesic pro-drug used clinically to control moderate pain with a
30
high analgesic efficacy and low toxicity. Dipyrone is hydrolyzed to 4-
31
methylaminoantipyrine (4-MAA), which is metabolized to 4-aminoantipyrine (4-AA).
32
Here, were investigate the involvement of peripheral cannabinoid CB2 and opioid
33
receptor activation in the local antihyperalgesic effect of dipyrone and 4-MAA. The
34
inflammatory agent, carrageenan was administered to the hindpaw of male Wistar
35
rats, and the mechanical nociceptive threshold was quantified by electronic von
36
Frey test. Dipyrone or 4-MAA were locally administered 2.5 h after carrageenan.
37
Following dipyrone injection, hindpaw tissue was harvested and its hydrolysis to 4-
38
MAA was analyzed by mass spectrometry (MS). The selective CB2 receptor
39
antagonist (AM630), naloxone (a non-selective opioid receptor antagonist), nor-BNI
40
(a selective kappa-opioid receptor), CTOP (a selective mu-opioid receptor), or
41
naltrindole (a selective delta-opioid receptor) was administered 30 min prior to 4-
42
MAA. The results demonstrate that carrageenan-induced mechanical hyperalgesia
43
was inhibited by dipyrone or 4-MAA in a dose-dependent manner. Dipyrone
44
administered to the hindpaw was completely hydrolyzed to 4-MAA. The
45
antihyperalgesic effect of 4-MAA was completely reversed by AM630, naloxone and
46
nor-BNI, but not by CTOP or naltrindole. These data suggest that the local
47
analgesic effect of dipyrone is mediated by its hydrolyzed bioactive form, 4-MAA
48
and, at least in part, depends on CB2 receptor and kappa-opioid receptor activation.
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In conclusion, the analgesic effect of dipyrone may involve a possible interaction
50
between the cannabinoid and opioid system in peripheral tissue.
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Keywords: Dipyrone; 4-methylaminoantipyrine; cannabinoid receptors; opioid
52
receptor.
2
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1.
Introduction
54 55
Dipyrone (metamizole) is an analgesic drug that has been used in clinical practice in
56
some countries for more than ten decades as it promotes a suitable analgesic effect
57
with very low toxicity. However, its analgesic mechanism of action is not completely
58
understood. As a pro-drug, dipyrone is characterized by fast hydrolysis to 4-
59
methylaminoantipyrine (4-MAA) (Pierre et al., 2007), which is then metabolized in
60
the liver to 4-formylaminoantipyrine (4-FAA), 4-aminoantipyrine (4-AA), and 4-
61
acetylaminoantipyrine (4-AAA). After oral administration of dipyrone, two
62
metabolites are found in human plasma and cerebrospinal fluid with the same
63
analgesic effect as dipyrone, 4-MAA and 4-AA, which are also known as dipyrone
64
bioactive metabolites (Pierre et al., 2007; Rogosch et al., 2012). Dipyrone was
65
initially considerate a non-steroidal anti-inflammatory drug (NSAID) (Frölich et al.,
66
1986; Lüthy et al., 1983). However, further studies have demonstrated that dipyrone
67
decreases prostaglandin synthesis only at higher doses than those necessary for
68
analgesic effect (Lorenzetti and Ferreira, 1985). In contrast to NSAIDs, dipyrone
69
decreases the hyperalgesia induced by prostaglandin E2 (PGE2), an inflammatory
70
mediator synthetized by COX enzyme activation that promotes hyperalgesia via
71
sensitization of nociceptors (Lorenzetti and Ferreira, 1985).
72 73
We have recently demonstrated that much like Dipyrone, 4-MAA, induces
74
antihyperalgesia through activation of the L-argenine-NO-cGMP-KATP pathway (Dos
75
Santos et al., 2014). Moreover, recent studies suggest involvement of cannabinoid
76
and opioid receptor activation in the analgesic effect of dipyrone (Rogosch et al.,
77
2012; Silva et al., 2016; Vazquez et al., 2005).
3
78
Cannabinoid receptors, CB1 and CB2, are G-protein-coupled receptors (GPCRs).
79
CB1 receptors are expressed primarily in the central and peripheral nervous
80
systems and CB2 receptors are mostly expressed in immune cells and keratinocytes
81
(Ahluwalia et al., 2000; Maione et al., 2015; Yang et al., 2013). The antihyperalgesic
82
effect of CB1 receptors is mediated by its own activation on the nociceptor
83
(Ahluwalia et al., 2000). On the other hand, the antihyperalgesic effect of CB2
84
receptor activation is thought to act via the release of endogenous opioids
85
(Ahluwalia et al., 2000; Ashton and Glass, 2007).
86 87
Likewise, opioids receptors are a group of G protein-coupled receptors expressed in
88
central and peripheral nervous systems (Ji et al., 1995). Delta (δ), kappa (κ), and
89
mu (µ) opioid receptors can be activated by the endogenous opioids encephalin,
90
dynorphin and endorphin, respectively. Moreover, an interaction between
91
cannabinoid and opioid systems has been shown to have analgesic effect in several
92
models of pain (Machado et al., 2014; Negrete et al., 2011). In fact, behavioral and
93
molecular studies have demonstrated that activation of CB2 receptors, induced
94
release of dynorphin-A and subsequent kappa-opioid receptor activation (Machado
95
et al., 2014).
96 97
Of the many neurochemical systems involved in the inhibitory control of pain, the
98
opioid and cannabinoid systems represent particular interest in terms of
99
physiological relevance and represent great targets for relevant therapeutic
100
approaches (Nadal et al., 2013). As such, the aim of this study was to investigate
101
the involvement of peripheral cannabinoid CB2 and opioid receptors in the analgesic
102
effects of dipyrone and 4-MAA.
4
103
2.
Materials and methods
104
2.1
Animals
105
A total of 180 male Wistar rats (Rattus norvegicus), weighing 150-250 g and 4-6-
106
weeks-old, were obtained from the Multidisciplinary Center for Biological Research
107
(CEMIB-UNICAMP, SP, Brazil). Experimental protocols were approved by Ethics
108
Committee for Animal Research of the University of Campinas (CEUA – UNICAMP,
109
protocol number: 3372-1). The animals were housed in plastic cages with food
110
(commercial chow for rodents) and filtered water available ad libitum. Testing
111
sessions took place during the light phase (09:00 AM - 5:00 PM) in a quiet room
112
maintained at 23º C. All experiments were conducted according to the IASP
113
guidelines for the use of laboratory animals (Zimmermann, 1983) and the Brazilian
114
Society of Laboratory Animal Science (SBCAL). All efforts were made to minimize
115
both stress and the number of animals necessary.
116 117
2.2
Drugs and doses
118
The following drugs were used: carrageenan (100 µg/paw); AM630, a selective
119
cannabinoid CB2 receptor antagonist (16.6, 50, and 150 µg/paw) (Machado et al.,
120
2014; Silva et al., 2012), AM251 a selective cannabinoid CB1 receptor antagonist
121
(80 and 240 µg/paw; Dos Santos et al., 2014); (dipyrone (8, 80, 160, and 320
122
µg/paw) (Romero and Duarte, 2013; Vivancos et al., 2004); 4-MAA (4-
123
methylaminoantipyrine; 8, 80, 160, and 320 µg/paw) (Dos Santos et al., 2014);
124
Naloxone, a non-selective opioid receptor antagonist (2, 10, and 20 µg/paw)
125
(Katsuyama et al., 2013; Rahn et al., 2010); nor-BNI, a selective κappa-opioid
126
receptor antagonist (5, 10, and 50 µg/paw) (Silva et al., 2016; Zambelli et al., 2014);
127
Naltrindole (N115 Sigma), a selective delta-opioid receptor (1, 3, and 9 µg/paw)
5
128
(Izquierdo et al., 2013); CTOP, a selective mu-opioid receptor antagonist (8, 20, and
129
32 µg/paw) (Zambelli et al., 2014). AM251, AM630 and Naltrindole were dissolved
130
in propylene glycol and 10 % DMSO to a concentration of 50 µg/µL and then
131
resuspended in propylene glycol to the working final concentration (about 1 %
132
DMSO). CTOP, Nor-BNI, Naloxone, dipyrone and carrageenan was dissolved in
133
saline. All drugs were obtained from Sigma-Aldrich (MO, USA), except 4-MAA,
134
which was obtained from TLC-USA. The administration of carrageenan (100
135
µg/paw) induces mechanical hyperalgesia that reaches its maximum response 3 h
136
after injection (McCarson, 2015; Winter et al., 1962). Mechanical nociceptive
137
threshold was tested 3 h after carrageenan administration, and all tested drugs
138
were administrated 30 min prior to testing (2.5 h after carrageenan injections)
139 140
2.3
Subcutaneous injection
141
Drugs or vehicle were injected subcutaneously into the rat hindpaw (plantar surface)
142
with aid of a BD Ultra-Fine® (30 gauge) insulin needles. The animals were quickly
143
contained and the volume of 50 µL was injected.
144 145
2.4
Nociceptive paw electronic pressure-meter test: von Frey test
146
Rats were placed in acrylic cages (12 × 20 × 17 cm) with a wire grid floor 15–30 min
147
before testing to acclimate. During this adaptation period, the paws were tested
148
around 2–3 times. The test consisted of inducing a hindpaw flexion reflex with a
149
hand-held force transducer (Insight, Ribeirao Preto – Brazil) coupled with a 0.5 mm2
150
polypropylene tip. Below the grid, a tilted mirror provided a clear view of the rat’s
151
hindpaw. The investigator applied the tip between the five distal footpads gradually
152
increasing the pressure. The pressure (calibrated in grams) was automatically
6
153
recorded when the paw was withdrawn. The stimulus was repeated six times and
154
the average of the three closest measures were used to calculate the withdrawal
155
threshold. The hyperalgesia is presented as ∆ (delta) withdrawal threshold (intensity
156
of hyperalgesia), by subtracting the basal values (before injections) from those
157
obtained after treatment. Rats which did not present a consistent response were not
158
included.
159 160 161
2.5
Mass spectrometry 2.5.1 HESI-MS
162
High Performance Liquid Chromatography (HPLC)-grade methanol was purchased
163
from Burdick & Jackson (Muskegon, MI). Dipyrone solution was diluted in methanol
164
(1:1000) and injected with 3.0 µL min-1 of flow rate on a Q-ExactiveTM (Thermo
165
Scientific, Germany) mass spectrometer with orbitrap analyzer, via ESI. The
166
experiments were performed in the positive mode.
167
2.5.2 DESI-MSI
168
Frozen tissues were sliced using a Leica CM 1900 cryostat microtome (Leica
169
Biosystems, Nussloch, Germany) under –20 °C, and each slice was cut at a
170
thickness of 14 µm. Tissue sections were transferred by thaw mounting onto
171
conventional microscope glass slides without any surface treatment and were
172
stored at −80 °C until the time of analysis. The analyses were performed in a Q-
173
ExactiveTM (Thermo Scientific, Germany), a hybrid Quadrupole-Orbitrap mass
174
spectrometer with a resolution of 75,000 at m/z 400 coupled with a source DESI-2D
175
platform of Prosolia® (model OS-3201) for data acquisition. Data were converted in
176
image by Firefly v.1.3.0.0. The images were edited and analyzed using the BioMAP
177
software (version 3.8.04). The DESI configuration was optimized at 55° spray angle,
7
178
5.0 kV spray voltage, 160 psi N2 nebulizing gas pressure and a sprayed solvent of
179
methanol (HPLC-grade) at a 1.5 µL min−1 flow rate, in positive mode. Images were
180
collected from m/z 100−1200 with a step sized of 200 µm, a scan rate of 740 µm
181
s−1, and a pixel size of 200 µm × 200 µm.
182 183
2.6
Statistical analysis
184
To determine if there were differences between groups, one-way ANOVA followed
185
by Tukey’s Multiple Comparison Test, or unpaired t-test was performed, and P-
186
values < 0.05 were considered statistically significant. All statistical analyses were
187
performed using the GraphPad Prism v7.00 for Windows (GraphPad Software). The
188
results were presented as the mean ± S.E.M of six rats per group.
189 190
3
Results
191 192
3.1 Local administration of dipyrone or 4-methylaminoantipyrine (4-MAA) in
193
peripheral tissue reduces the carrageenan-induced hyperalgesia.
194
Local, subcutaneous, administration of carrageenan (100 µg/paw) into the
195
hindpaw induced mechanical hyperalgesia in rats, measured 3 h after injection.
196
Dipyrone or 4-MAA (all 8; 80; 160 or 320 µg/paw) administered to the same site 2.5
197
h after carrageenan decreased mechanical hyperalgesia in a dose-dependent
198
manner (one-way ANOVA followed by Tukey’s test; 1A: F7,40 = 26.93; 1B: F7,40 =
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33.83; P < 0.05) (Fig. 1A-B). However, administration of dipyrone (160 µg/paw) or
200
4-MAA (160 µg/paw) to the contralateral hindpaw did not change the mechanical
201
withdrawal threshold, ruling out a systemic effect.
202
8
203
3.2 Dipyrone is hydrolysate to 4-MAA in the hindpaw.
204
Because we observed the same action of Dipyrone and 4-MAA at the same dose,
205
we performed an assay to analyze whether dipyrone is metabolized to 4-MAA
206
locally in peripheral tissue using mass spectrometry. We observed two peaks
207
following Dipyrone administration, the first at m/z = 218.12843 (1.65 ppm of error),
208
and the second m/z = 240.11021 (2.16 ppm of error) (Fig. 2A). These ions were
209
assigned to the product of hydrolysis of the dipyrone, the 4-MAA [4-MAA+H]+ and
210
the adduct of the 4-MAA with Na [4-MAA+Na]+, respectively. No signal of dipyrone
211
(MW: 333 g/mol) was found in the mass spectrum, neither protonated nor sodiated.
212
Alkaline solution administration was used as a control. This result demonstrated that
213
dipyrone was hydrolyzed to 4-MAA, when solubilized in water.
214 215
As shown in Fig. 2 (panels B and C), the distribution of 4-MAA in hindpaw
216
subcutaneous tissue after dipyrone administration was analyzed by DESI-MSI.
217
Dipyrone administration in the subcutaneous tissue of hindpaw was distributed in [4-
218
MAA+H]+ (Fig. 2B) and [4-MAA+Na]+ (Fig. 2C) in a similar tissue area. No dipyrone
219
itself was found distributed in hindpaw tissue.
220 221
3.3 The antihyperalgesic effect of 4-methylaminoantipyrine is mediated by
222
CB2 and opioid receptor activation.
223
Because only 4-MAA was found in the peripheral tissue after local administration of
224
dipyrone, the following experiments were performed using only 4-MAA. To analyze
225
the involvement of opioid and cannabinoid CB2 receptors in the 4-MAA analgesic
226
effect, the non-selective opioid antagonist (Naloxone), and the selective
227
cannabinoid CB2 receptor antagonist (AM630) were administered 30 min before 4-
9
228
MAA. Local administration of carrageenan (100 µg/paw) in subcutaneous tissue of
229
rat hindpaw induced mechanical hyperalgesia that was reduced by 4-MAA (160
230
µg/paw). As shown in Fig. 3 (A and B) 4-MAA’s analgesic effect is reversed by
231
Naloxone (10, and 20 µg/paw) and AM630 (50, and 150 µg/paw) (one-way ANOVA
232
followed by Tukey’s test; 3A: F6,35 = 42.87; P < 0.05; 3B: F6,35 = 35.94;).
233
Because CB1 receptors have been shown to be involved in dipyrone’s analgesic
234
effect in PGE2 induced hyperalgesia, we used the CB1 antagonist, AM251, in two
235
effective doses (80 and 240 µg/paw, Dos Santos et al., 2014) to analyze the
236
involvement of CB1 receptor in carrageenan induced hyperalgesia. AM251 (80 and
237
240 µg/paw) did not affect the analgesic action of 4-MAA (Fig. 3C; one-way ANOVA
238
followed by Tukey’s test; F4,23 = 156; P > 0.05). The administration of the naloxone,
239
vehicle, or AM630 alone did not change the mechanical withdraw threshold
240
following carrageenan (one-way ANOVA followed by Tukey’s test, P > 0.05).
241 242
3.4 The antihyperalgesic effect of 4-methylaminoantipyrine is mediated by
243
kappa-opioid receptor activation.
244
Because 4-MAA’s analgesic effect was reversed by the non-selective opioid
245
antagonist naloxone, we sought to determine which opioid receptor(s) play a role in
246
the effects of Dipyrone. To analyze the involvement of kappa-, mu-, and delta-opioid
247
receptors in the analgesic effect of 4-MAA, selective antagonists of these receptors
248
were administered 30 min before 4-MAA. As shown in Fig. 4 (panel A), the
249
pretreatment with nor-BNI, a selective kappa-opioid receptor antagonist (5, 10 or 50
250
µg/paw), reversed the analgesic effect of 4-MAA in a dose-dependent manner (one-
251
way ANOVA followed by Tukey’s test; F5,30 = 46.29; P < 0.05). However, CTOP, a
252
selective mu-opioid receptor antagonist (Fig. 4B; 8, 20, and 32 µg/paw), and
10
253
Naltrindole, a selective delta-opioid receptor antagonist (Fig. 4C; 1, 3, and 9
254
µg/paw), both administered 30 min before 4-MAA injection did not reverse 4-MAA’s
255
analgesic effect (one-way ANOVA followed by Tukey’s test; 4B: F5,30 = 22.97; 4C:
256
F5,30 = 15.05; P > 0.05).
257 258
4 . Discussion
259 260
We have recently demonstrated that dipyrone and its bioactive metabolite 4-MAA
261
have a similar analgesic effect, and both induced activation of L-argenine-NO-KATP
262
pathway (Dos Santos et al., 2014). In the current study, we showed that dipyrone is
263
quickly hydrolyzed to 4-MAA in peripheral tissue, suggesting that dipyrone’s
264
analgesic effect could be mediated by 4-MAA. We also showed that 4-MAA’s
265
analgesic effect is dependent on CB2 and kappa-opioid receptor activation.
266 267
Our results demonstrated that dipyrone or 4-MAA, locally administered in peripheral
268
tissue, inhibit the carrageenan-induced hyperalgesia, which corroborate with
269
previous studies that have demonstrated dipyrone’s analgesic effect in various pain
270
models (Beirith et al., 1998; Dos Santos et al., 2014; Edwards et al., 2010; Rogosch
271
et al., 2012; Vazquez et al., 2005). Carrageenan is an inflammatory agent that
272
induces hyperalgesia by release of endogenous prostaglandins, especially PGE2,
273
which ultimately sensitizes the nociceptors (Lorenzetti and Ferreira, 1985).
274 275
Although dipyrone is metabolized into two bioactive compounds, 4-MAA and 4-AA,
276
this study demonstrated that either dipyrone or 4-MAA prevented the hyperalgesia
277
induced by the inflammatory agent carrageenan, suggesting that the metabolite 4-
11
278
MAA alone is enough to induce effective analgesic effect despite 4-AA.
279
Furthermore, dipyrone was completely hydrolyzed to 4-MAA in the peripheral tissue,
280
confirming our hypotheses that dipyrone’s analgesic effect is mediated by local
281
hydrolysis to 4-MAA (Dos Santos et al., 2014). In fact, some studies have shown
282
that dipyrone’s hydrolysis to 4-MAA is a non-enzymatic reaction that depends on
283
concentration, pH, and temperature (Cohen et al., 1998; Pierre et al., 2007).
284 285
The findings of this study also demonstrated after local dipyrone administration the
286
metabolite 4-MAA can be detected in the peripheral tissue in two forms: protonated
287
[4-MAA+H]+ and sodiated [4-MAA+Na]+. The solution analyzed by HESI before
288
local administration in the peripheral tissue showed a higher sodium concentration
289
(sodiated adduct) when compared with the same solution administrated in the tissue
290
(protonated adduct). This lower concentration of sodium in the tissue and higher of
291
proton is probably because sodium solubilized throughout the tissue creating a
292
lower ionization of this particular ion form. However, independently of the form that
293
the molecule was detected, sodiated or protonated, the results demonstrated that
294
the only product detected was 4-MAA.
295 296
Our results have also shown that 4-MAA’s antihyperalgesic effect depends on CB2
297
cannabinoid receptor activation and kappa-opioid receptors activation. Although
298
CB2 receptor activation can modulate the immune response in peripheral tissue
299
(Lunn et al., 2006), data of this study strongly suggests that 4-MAA decreases the
300
inflammatory hyperalgesia through releasing endogenous opioids followed by
301
kappa-opioid receptor activation. There is robust data showing that CB2 receptor
302
activation induces analgesia by releasing endogenous opioids, which then
12
303
hyperpolarize peripheral nociceptors (Negrete et al., 2011). Therefore, it is plausible
304
to hypothesize that the antihyperalgesic effect of dipyrone in the peripheral tissue is
305
not associated with an anti-inflammatory effect but with the release of endogenous
306
opioids. Nevertheless, because CB2 receptor is probably not expressed in
307
peripheral nociceptors (Freund et al., 2003), the analgesic effect of dipyrone, or
308
more specifically 4-MAA in peripheral tissue may depend on migration of
309
inflammatory cells. These data may explain previous results from our laboratory,
310
where we showed that the analgesic effect of dipyrone or 4-MAA in PGE2-induced
311
hyperalgesia does not depend of CB2 receptor activation (Dos Santos et al., 2014).
312
However, with the carrageenan-induced hyperalgesia model, the analgesic effect of
313
4-MAA was completely prevented by a CB2 receptor antagonist. This difference
314
could be explained by the fact that PGE2 (100 ng) does not induce cell migration to
315
the same degree as carrageenan (Cunha et al., 2008).
316 317
A crosstalk between cannabinoid and opioid systems in pain modulation has been
318
suggested (Machado et al., 2014; Negrete et al., 2011; Guida et al., 2012). Although
319
it is still unclear the mechanism of this interaction, the crosstalk between these two
320
systems has been shown to reduce acute inflammatory or chronic pain (Machado et
321
al., 2014;Guida et al., 2012). Indeed, salvinorin’s analgesic effect depends of CB1
322
and kappa-opioid receptor (KOR), moreover a synthetic KOR agonist is
323
counteracted by AM251, a CB1 antagonist, suggesting a crosstalk between
324
KOR/CB1 receptor (Guida et al., 2012). Also, significant data has been published
325
demonstrating a functional relationship between CB2/ KOR, in fact CB2 receptor
326
activation has been shown to induce endogenous opioid releasing triggering KOR
327
receptor activation (Machado et al., 2014; Negrete et al., 2011).
13
328 329 330
The endocannabinoid system is composed of endogenous ligands anandamide
331
(AEA, arachidonoylethanolamide) and 2-arachidonoylglycerol (2-AG) and
332
cannabinoid CB1 and CB2 receptors, expressed in central and peripheral nervous
333
systems. Whereas data of this study demonstrated that the antihyperalgesic effect
334
of 4-MAA is reversed by AM630, a selective CB2 receptor antagonist, this study
335
does not demonstrate the mechanism underlying CB2 receptor activation by 4-MAA.
336
Indeed, recent data has shown that 4-MAA and 4-AA are a precursor of
337
arachidonoyl amides that induces stimulation of CB1 and CB2 receptors (Rogosch et
338
al., 2012). Data from our laboratory broaden this perspective, showing that the
339
analgesic effect of 4-MAA depends on CB2 receptor activation. Moreover, 4-MAA
340
analgesic effect does not involve CB1 receptor activation in carrageenan-induced
341
pain corroborating our previous, which used prostaglandin as model of pain (Dos
342
Santos et al., 2014). Although carrageenan and prostaglandin induce pain in
343
different ways, carrageenan overlap PGE2 as a final inflammatory mediator that
344
sensitize the nociceptors (Lorenzetti and Ferreira, 1985). These data reinforce that
345
4-MAA induced analgesia independently of the CB1 receptor. On the other hand, 4-
346
AA’s analgesic effect is mediated by CB1 receptor when its antihyperalgesic effect is
347
prevented by the administration of AM251 a selective cannabinoid CB1 receptor
348
antagonist (Dos Santos et al., 2014).
349 350
Because this study demonstrated the involvement of kappa-opioid receptor, but not
351
mu or delta opioid receptors, it is possible that the endogenous opioid released by
352
CB2 receptor activation after 4-MAA is dynorphin. Previous data from our laboratory
14
353
demonstrated that the administration of supernatant of primary culture of
354
keratinocytes or fibroblasts blocks the hyperalgesia induced by carrageenan in rat
355
hind paw, which is prevented by mu, but not by delta or kappa opioid receptor
356
antagonists. These data may rule out the involvement of these cells in the
357
antihyperalgesic effect of 4-MAA. On the other hand, it has been demonstrated that
358
adoptive transfer of M2, but not M0 or M1 macrophage, alleviates the inflammatory
359
hyperalgesia by release of endogenous opioids, including dynorphin (Pannell et al.,
360
2016). Future studies in our laboratory will address the involvement of macrophages
361
in the antihyperalgesic effect of dipyrone and its metabolites. In agreeing with our
362
results, it was recently demonstrated that crotalphine, a non-opioid peptide, induces
363
antihyperalgesic effect dependent on the peripheral CB2 cannabinoid receptor
364
activation and the subsequent release of dynorphin (Machado et al., 2014).
365 366
In conclusion, data of this study suggest a new mechanism of action for dipyrone as
367
well as 4-MAA, which involves an interaction between peripheral CB2 and opioid
368
systems. This interaction represents a new strategy to treat inflammatory pain
369
without inducing central nervous system-mediated undesirable effects and point to
370
4-MAA as a bioactive metabolite and new therapeutic for the treatment of
371
inflammatory pain with minimal side-effects.
372 373 374 375
Acknowledgements This work was funded by CAPES (Coordination of Improvement of Higher Education Personnel).
376 377
Conflict of interest
15
378
The authors declare no conflict of interest.
379 380
Fig.s Legends
381
Fig. 1. Local administration of dipyrone or 4-methylaminoantipyrine (4-MAA) in
382
peripheral tissue reduces the carrageenan-induced hyperalgesia. Dipyrone (Dipy) or
383
4-MAA administered in the hindpaw 2.5 h after carrageenan inhibited, in a dose-
384
dependent manner, the mechanical hyperalgesia evaluated 30 min later
385
(respectively Fig. 1A, 1B). Dipyrone or 4-MAA (160 µg/paw, but not 320 µg/paw),
386
administered in contra-lateral paw (Ct) did not change the mechanical withdrawal
387
threshold, ruling out its systemic effect. The symbol “*” means different from control
388
group (0.9% NaCl administration; 50 µL), “#” means different from carrageenan
389
group (one-way ANOVA followed by Tukey’s test; P < 0.05).
390 391
Fig. 2. Dipyrone is hydrolysate to 4-MAA in the hindpaw. (2A) In Mass spectrum the
392
presence of two peaks can be noticed, the first at 218 m/z (1.65 ppm) and the
393
second 240 m/z (2.16 ppm). These peaks were assigned to the molecular peak of
394
4-MAA [4-MAA+H]+ and the adduct of the 4-MAA with Na, [4-MAA+Na]+
395
respectively. The dipyrone administration in the subcutaneous tissue of hindpaw
396
distributed in [4- MAA+H]+ and [4-MAA+Na]+ (Fig. 2B and 2C, respectively) in a
397
similar tissue area when analyzed by DESI-MSI. No signal of dipyrone (MW: 333
398
g/mol) was found in the mass spectrum.
399 400
Fig. 3. Antihyperalgesic effect of 4-methylaminoantipyrine (4-MAA) is mediated by
401
CB2 and opioid receptor activation. Local administration of carrageenan in rat´s
402
hindpaw induced mechanical hyperalgesia 3 h after, which is decreased with 4-MAA
16
403
administered 2.5 h after carrageenan. This antihyperalgesic effect was reversed by
404
Naloxone (3A 2, 10 and 20 µg/paw) or AM630 (3B 50 and 150 µg/paw) but not by
405
AM251 (3C 80 and 240 µg/paw). The symbol “*” means different from control group
406
(0.9% NaCl; 50 µL), “#” means different from carrageenan group and “&” means
407
different from carrageenan plus 4-MAA (one-way ANOVA followed by Tukey’s test;
408
P < 0.05). The vehicle’s administration of 4-MAA or AM630 (propylene glycol+0.1%
409
DMSO) does not change the mechanical withdraw threshold by itself.
410 411
Fig. 4. Antihyperalgesic effect of 4-methylaminoantipyrine (4-MAA) is mediated by
412
kappa-opioid receptor activation. Local administration of carrageenan in rat´s
413
hindpaw induced mechanical hyperalgesia 3 h after, which is decreased with 4-MAA
414
administered 2.5 h after carrageenan. This antihyperalgesic effect was reversed by
415
nor-BNI, a selective Kappa-opioid receptor antagonist (4A 5, 10 and 50 µg/paw) but
416
not CTOP, a selective Mu-opioid receptor antagonist (4B 8, 20 and 32 µg/paw) or
417
Naltrindole, a selective delta-opioid receptor antagonist (4C 1, 3 and 9 µg/paw). The
418
symbol “*” means different from control group (0.9% NaCl; 50 µL), “#” means
419
different from carrageenan group (one-way ANOVA followed by Tukey’s test; P <
420
0.05). The administration of vehicle does not change the mechanical withdraw
421
threshold by itself.
422 423
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S0014-2999(20)30097-2
DOI:
https://doi.org/10.1016/j.ejphar.2020.173005
Reference:
EJP 173005
To appear in:
European Journal of Pharmacology
Received Date: 26 November 2019 Revised Date:
3 February 2020
Accepted Date: 7 February 2020
Please cite this article as: Gonçalves dos Santos, G., Vieira, W.F., Vendramini, P.H., Bassani da Silva, B., Magalhães, S.F., Tambeli, Clá.Herrera., Parada, C.A., Dipyrone is locally hydrolyzed to 4methylaminoantipyrine and its antihyperalgesic effect depends on CB2 and kappa-opioid receptors activation, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/j.ejphar.2020.173005. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Dipyrone is locally hydrolyzed to 4-methylaminoantipyrine and its antihyperalgesic effect depends on CB2 and kappa-opioid receptors activation.
Gilson Gonçalves dos Santos¹*; Willians Fernando Vieira¹; Pedro Henrique Vendramini2; Bianca Bassani da Silva¹, Silviane Fernandes Magalhães¹; Cláudia Herrera Tambeli¹; Carlos Amilcar Parada¹
¹Department of Structural and Functional Biology, Institute of Biology, University of Campinas (UNICAMP), ZIP Code 13083-864, Campinas, Sao Paulo, Brazil; 2
ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of
Campinas (UNICAMP), ZIP Code 13083-970, Campinas, Sao Paulo, Brazil.
*Corresponding author: [email protected]. Full postal address: Department of Anesthesiology, University of California, San Diego, 9500 Gilman Drive, Mail Code 0818, La Jolla, CA 92093-0818
1 2 3
Dipyrone is locally hydrolyzed to 4-methylaminoantipyrine and its antihyperalgesic effect depends on CB2 and kappa-opioid receptors activation.
4 5
Gilson Gonçalves dos Santos¹*; Willians Fernando Vieira¹;
6
Pedro Henrique Vendramini2; Bianca Bassani da Silva¹, Silviane Fernandes
7
Magalhães; Cláudia Herrera Tambeli¹; Carlos Amilcar Parada¹
8
¹Department of Structural and Functional Biology, Institute of Biology, University of
9
Campinas (UNICAMP), ZIP Code 13083-864, Campinas, Sao Paulo, Brazil;
10
2
11
Campinas (UNICAMP), ZIP Code 13083-970, Campinas, Sao Paulo, Brazil.
ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of
12 13
*Corresponding author: [email protected]. Full postal
14
address: Department of Anesthesiology, University of California, San Diego, 9500
15
Gilman Drive, Mail Code 0818, La Jolla, CA 92093-0818
16
Co-author’s e-mail addresses:
17
W.F.V.: [email protected]
18
P.H.V.: [email protected]
19
B.B.S.: [email protected]
20 21
S.F.M.: [email protected]
22
C.H.T.: [email protected] C.A.P.: [email protected]
23 24 25
Acknowledgements
26 27
1
28
Abstract
29
Dipyrone is an analgesic pro-drug used clinically to control moderate pain with a
30
high analgesic efficacy and low toxicity. Dipyrone is hydrolyzed to 4-
31
methylaminoantipyrine (4-MAA), which is metabolized to 4-aminoantipyrine (4-AA).
32
Here, were investigate the involvement of peripheral cannabinoid CB2 and opioid
33
receptor activation in the local antihyperalgesic effect of dipyrone and 4-MAA. The
34
inflammatory agent, carrageenan was administered to the hindpaw of male Wistar
35
rats, and the mechanical nociceptive threshold was quantified by electronic von
36
Frey test. Dipyrone or 4-MAA were locally administered 2.5 h after carrageenan.
37
Following dipyrone injection, hindpaw tissue was harvested and its hydrolysis to 4-
38
MAA was analyzed by mass spectrometry (MS). The selective CB2 receptor
39
antagonist (AM630), naloxone (a non-selective opioid receptor antagonist), nor-BNI
40
(a selective kappa-opioid receptor), CTOP (a selective mu-opioid receptor), or
41
naltrindole (a selective delta-opioid receptor) was administered 30 min prior to 4-
42
MAA. The results demonstrate that carrageenan-induced mechanical hyperalgesia
43
was inhibited by dipyrone or 4-MAA in a dose-dependent manner. Dipyrone
44
administered to the hindpaw was completely hydrolyzed to 4-MAA. The
45
antihyperalgesic effect of 4-MAA was completely reversed by AM630, naloxone and
46
nor-BNI, but not by CTOP or naltrindole. These data suggest that the local
47
analgesic effect of dipyrone is mediated by its hydrolyzed bioactive form, 4-MAA
48
and, at least in part, depends on CB2 receptor and kappa-opioid receptor activation.
49
In conclusion, the analgesic effect of dipyrone may involve a possible interaction
50
between the cannabinoid and opioid system in peripheral tissue.
51
Keywords: Dipyrone; 4-methylaminoantipyrine; cannabinoid receptors; opioid
52
receptor.
2
53
1.
Introduction
54 55
Dipyrone (metamizole) is an analgesic drug that has been used in clinical practice in
56
some countries for more than ten decades as it promotes a suitable analgesic effect
57
with very low toxicity. However, its analgesic mechanism of action is not completely
58
understood. As a pro-drug, dipyrone is characterized by fast hydrolysis to 4-
59
methylaminoantipyrine (4-MAA) (Pierre et al., 2007), which is then metabolized in
60
the liver to 4-formylaminoantipyrine (4-FAA), 4-aminoantipyrine (4-AA), and 4-
61
acetylaminoantipyrine (4-AAA). After oral administration of dipyrone, two
62
metabolites are found in human plasma and cerebrospinal fluid with the same
63
analgesic effect as dipyrone, 4-MAA and 4-AA, which are also known as dipyrone
64
bioactive metabolites (Pierre et al., 2007; Rogosch et al., 2012). Dipyrone was
65
initially considerate a non-steroidal anti-inflammatory drug (NSAID) (Frölich et al.,
66
1986; Lüthy et al., 1983). However, further studies have demonstrated that dipyrone
67
decreases prostaglandin synthesis only at higher doses than those necessary for
68
analgesic effect (Lorenzetti and Ferreira, 1985). In contrast to NSAIDs, dipyrone
69
decreases the hyperalgesia induced by prostaglandin E2 (PGE2), an inflammatory
70
mediator synthetized by COX enzyme activation that promotes hyperalgesia via
71
sensitization of nociceptors (Lorenzetti and Ferreira, 1985).
72 73
We have recently demonstrated that much like Dipyrone, 4-MAA, induces
74
antihyperalgesia through activation of the L-argenine-NO-cGMP-KATP pathway (Dos
75
Santos et al., 2014). Moreover, recent studies suggest involvement of cannabinoid
76
and opioid receptor activation in the analgesic effect of dipyrone (Rogosch et al.,
77
2012; Silva et al., 2016; Vazquez et al., 2005).
3
78
Cannabinoid receptors, CB1 and CB2, are G-protein-coupled receptors (GPCRs).
79
CB1 receptors are expressed primarily in the central and peripheral nervous
80
systems and CB2 receptors are mostly expressed in immune cells and keratinocytes
81
(Ahluwalia et al., 2000; Maione et al., 2015; Yang et al., 2013). The antihyperalgesic
82
effect of CB1 receptors is mediated by its own activation on the nociceptor
83
(Ahluwalia et al., 2000). On the other hand, the antihyperalgesic effect of CB2
84
receptor activation is thought to act via the release of endogenous opioids
85
(Ahluwalia et al., 2000; Ashton and Glass, 2007).
86 87
Likewise, opioids receptors are a group of G protein-coupled receptors expressed in
88
central and peripheral nervous systems (Ji et al., 1995). Delta (δ), kappa (κ), and
89
mu (µ) opioid receptors can be activated by the endogenous opioids encephalin,
90
dynorphin and endorphin, respectively. Moreover, an interaction between
91
cannabinoid and opioid systems has been shown to have analgesic effect in several
92
models of pain (Machado et al., 2014; Negrete et al., 2011). In fact, behavioral and
93
molecular studies have demonstrated that activation of CB2 receptors, induced
94
release of dynorphin-A and subsequent kappa-opioid receptor activation (Machado
95
et al., 2014).
96 97
Of the many neurochemical systems involved in the inhibitory control of pain, the
98
opioid and cannabinoid systems represent particular interest in terms of
99
physiological relevance and represent great targets for relevant therapeutic
100
approaches (Nadal et al., 2013). As such, the aim of this study was to investigate
101
the involvement of peripheral cannabinoid CB2 and opioid receptors in the analgesic
102
effects of dipyrone and 4-MAA.
4
103
2.
Materials and methods
104
2.1
Animals
105
A total of 180 male Wistar rats (Rattus norvegicus), weighing 150-250 g and 4-6-
106
weeks-old, were obtained from the Multidisciplinary Center for Biological Research
107
(CEMIB-UNICAMP, SP, Brazil). Experimental protocols were approved by Ethics
108
Committee for Animal Research of the University of Campinas (CEUA – UNICAMP,
109
protocol number: 3372-1). The animals were housed in plastic cages with food
110
(commercial chow for rodents) and filtered water available ad libitum. Testing
111
sessions took place during the light phase (09:00 AM - 5:00 PM) in a quiet room
112
maintained at 23º C. All experiments were conducted according to the IASP
113
guidelines for the use of laboratory animals (Zimmermann, 1983) and the Brazilian
114
Society of Laboratory Animal Science (SBCAL). All efforts were made to minimize
115
both stress and the number of animals necessary.
116 117
2.2
Drugs and doses
118
The following drugs were used: carrageenan (100 µg/paw); AM630, a selective
119
cannabinoid CB2 receptor antagonist (16.6, 50, and 150 µg/paw) (Machado et al.,
120
2014; Silva et al., 2012), AM251 a selective cannabinoid CB1 receptor antagonist
121
(80 and 240 µg/paw; Dos Santos et al., 2014); (dipyrone (8, 80, 160, and 320
122
µg/paw) (Romero and Duarte, 2013; Vivancos et al., 2004); 4-MAA (4-
123
methylaminoantipyrine; 8, 80, 160, and 320 µg/paw) (Dos Santos et al., 2014);
124
Naloxone, a non-selective opioid receptor antagonist (2, 10, and 20 µg/paw)
125
(Katsuyama et al., 2013; Rahn et al., 2010); nor-BNI, a selective κappa-opioid
126
receptor antagonist (5, 10, and 50 µg/paw) (Silva et al., 2016; Zambelli et al., 2014);
127
Naltrindole (N115 Sigma), a selective delta-opioid receptor (1, 3, and 9 µg/paw)
5
128
(Izquierdo et al., 2013); CTOP, a selective mu-opioid receptor antagonist (8, 20, and
129
32 µg/paw) (Zambelli et al., 2014). AM251, AM630 and Naltrindole were dissolved
130
in propylene glycol and 10 % DMSO to a concentration of 50 µg/µL and then
131
resuspended in propylene glycol to the working final concentration (about 1 %
132
DMSO). CTOP, Nor-BNI, Naloxone, dipyrone and carrageenan was dissolved in
133
saline. All drugs were obtained from Sigma-Aldrich (MO, USA), except 4-MAA,
134
which was obtained from TLC-USA. The administration of carrageenan (100
135
µg/paw) induces mechanical hyperalgesia that reaches its maximum response 3 h
136
after injection (McCarson, 2015; Winter et al., 1962). Mechanical nociceptive
137
threshold was tested 3 h after carrageenan administration, and all tested drugs
138
were administrated 30 min prior to testing (2.5 h after carrageenan injections)
139 140
2.3
Subcutaneous injection
141
Drugs or vehicle were injected subcutaneously into the rat hindpaw (plantar surface)
142
with aid of a BD Ultra-Fine® (30 gauge) insulin needles. The animals were quickly
143
contained and the volume of 50 µL was injected.
144 145
2.4
Nociceptive paw electronic pressure-meter test: von Frey test
146
Rats were placed in acrylic cages (12 × 20 × 17 cm) with a wire grid floor 15–30 min
147
before testing to acclimate. During this adaptation period, the paws were tested
148
around 2–3 times. The test consisted of inducing a hindpaw flexion reflex with a
149
hand-held force transducer (Insight, Ribeirao Preto – Brazil) coupled with a 0.5 mm2
150
polypropylene tip. Below the grid, a tilted mirror provided a clear view of the rat’s
151
hindpaw. The investigator applied the tip between the five distal footpads gradually
152
increasing the pressure. The pressure (calibrated in grams) was automatically
6
153
recorded when the paw was withdrawn. The stimulus was repeated six times and
154
the average of the three closest measures were used to calculate the withdrawal
155
threshold. The hyperalgesia is presented as ∆ (delta) withdrawal threshold (intensity
156
of hyperalgesia), by subtracting the basal values (before injections) from those
157
obtained after treatment. Rats which did not present a consistent response were not
158
included.
159 160 161
2.5
Mass spectrometry 2.5.1 HESI-MS
162
High Performance Liquid Chromatography (HPLC)-grade methanol was purchased
163
from Burdick & Jackson (Muskegon, MI). Dipyrone solution was diluted in methanol
164
(1:1000) and injected with 3.0 µL min-1 of flow rate on a Q-ExactiveTM (Thermo
165
Scientific, Germany) mass spectrometer with orbitrap analyzer, via ESI. The
166
experiments were performed in the positive mode.
167
2.5.2 DESI-MSI
168
Frozen tissues were sliced using a Leica CM 1900 cryostat microtome (Leica
169
Biosystems, Nussloch, Germany) under –20 °C, and each slice was cut at a
170
thickness of 14 µm. Tissue sections were transferred by thaw mounting onto
171
conventional microscope glass slides without any surface treatment and were
172
stored at −80 °C until the time of analysis. The analyses were performed in a Q-
173
ExactiveTM (Thermo Scientific, Germany), a hybrid Quadrupole-Orbitrap mass
174
spectrometer with a resolution of 75,000 at m/z 400 coupled with a source DESI-2D
175
platform of Prosolia® (model OS-3201) for data acquisition. Data were converted in
176
image by Firefly v.1.3.0.0. The images were edited and analyzed using the BioMAP
177
software (version 3.8.04). The DESI configuration was optimized at 55° spray angle,
7
178
5.0 kV spray voltage, 160 psi N2 nebulizing gas pressure and a sprayed solvent of
179
methanol (HPLC-grade) at a 1.5 µL min−1 flow rate, in positive mode. Images were
180
collected from m/z 100−1200 with a step sized of 200 µm, a scan rate of 740 µm
181
s−1, and a pixel size of 200 µm × 200 µm.
182 183
2.6
Statistical analysis
184
To determine if there were differences between groups, one-way ANOVA followed
185
by Tukey’s Multiple Comparison Test, or unpaired t-test was performed, and P-
186
values < 0.05 were considered statistically significant. All statistical analyses were
187
performed using the GraphPad Prism v7.00 for Windows (GraphPad Software). The
188
results were presented as the mean ± S.E.M of six rats per group.
189 190
3
Results
191 192
3.1 Local administration of dipyrone or 4-methylaminoantipyrine (4-MAA) in
193
peripheral tissue reduces the carrageenan-induced hyperalgesia.
194
Local, subcutaneous, administration of carrageenan (100 µg/paw) into the
195
hindpaw induced mechanical hyperalgesia in rats, measured 3 h after injection.
196
Dipyrone or 4-MAA (all 8; 80; 160 or 320 µg/paw) administered to the same site 2.5
197
h after carrageenan decreased mechanical hyperalgesia in a dose-dependent
198
manner (one-way ANOVA followed by Tukey’s test; 1A: F7,40 = 26.93; 1B: F7,40 =
199
33.83; P < 0.05) (Fig. 1A-B). However, administration of dipyrone (160 µg/paw) or
200
4-MAA (160 µg/paw) to the contralateral hindpaw did not change the mechanical
201
withdrawal threshold, ruling out a systemic effect.
202
8
203
3.2 Dipyrone is hydrolysate to 4-MAA in the hindpaw.
204
Because we observed the same action of Dipyrone and 4-MAA at the same dose,
205
we performed an assay to analyze whether dipyrone is metabolized to 4-MAA
206
locally in peripheral tissue using mass spectrometry. We observed two peaks
207
following Dipyrone administration, the first at m/z = 218.12843 (1.65 ppm of error),
208
and the second m/z = 240.11021 (2.16 ppm of error) (Fig. 2A). These ions were
209
assigned to the product of hydrolysis of the dipyrone, the 4-MAA [4-MAA+H]+ and
210
the adduct of the 4-MAA with Na [4-MAA+Na]+, respectively. No signal of dipyrone
211
(MW: 333 g/mol) was found in the mass spectrum, neither protonated nor sodiated.
212
Alkaline solution administration was used as a control. This result demonstrated that
213
dipyrone was hydrolyzed to 4-MAA, when solubilized in water.
214 215
As shown in Fig. 2 (panels B and C), the distribution of 4-MAA in hindpaw
216
subcutaneous tissue after dipyrone administration was analyzed by DESI-MSI.
217
Dipyrone administration in the subcutaneous tissue of hindpaw was distributed in [4-
218
MAA+H]+ (Fig. 2B) and [4-MAA+Na]+ (Fig. 2C) in a similar tissue area. No dipyrone
219
itself was found distributed in hindpaw tissue.
220 221
3.3 The antihyperalgesic effect of 4-methylaminoantipyrine is mediated by
222
CB2 and opioid receptor activation.
223
Because only 4-MAA was found in the peripheral tissue after local administration of
224
dipyrone, the following experiments were performed using only 4-MAA. To analyze
225
the involvement of opioid and cannabinoid CB2 receptors in the 4-MAA analgesic
226
effect, the non-selective opioid antagonist (Naloxone), and the selective
227
cannabinoid CB2 receptor antagonist (AM630) were administered 30 min before 4-
9
228
MAA. Local administration of carrageenan (100 µg/paw) in subcutaneous tissue of
229
rat hindpaw induced mechanical hyperalgesia that was reduced by 4-MAA (160
230
µg/paw). As shown in Fig. 3 (A and B) 4-MAA’s analgesic effect is reversed by
231
Naloxone (10, and 20 µg/paw) and AM630 (50, and 150 µg/paw) (one-way ANOVA
232
followed by Tukey’s test; 3A: F6,35 = 42.87; P < 0.05; 3B: F6,35 = 35.94;).
233
Because CB1 receptors have been shown to be involved in dipyrone’s analgesic
234
effect in PGE2 induced hyperalgesia, we used the CB1 antagonist, AM251, in two
235
effective doses (80 and 240 µg/paw, Dos Santos et al., 2014) to analyze the
236
involvement of CB1 receptor in carrageenan induced hyperalgesia. AM251 (80 and
237
240 µg/paw) did not affect the analgesic action of 4-MAA (Fig. 3C; one-way ANOVA
238
followed by Tukey’s test; F4,23 = 156; P > 0.05). The administration of the naloxone,
239
vehicle, or AM630 alone did not change the mechanical withdraw threshold
240
following carrageenan (one-way ANOVA followed by Tukey’s test, P > 0.05).
241 242
3.4 The antihyperalgesic effect of 4-methylaminoantipyrine is mediated by
243
kappa-opioid receptor activation.
244
Because 4-MAA’s analgesic effect was reversed by the non-selective opioid
245
antagonist naloxone, we sought to determine which opioid receptor(s) play a role in
246
the effects of Dipyrone. To analyze the involvement of kappa-, mu-, and delta-opioid
247
receptors in the analgesic effect of 4-MAA, selective antagonists of these receptors
248
were administered 30 min before 4-MAA. As shown in Fig. 4 (panel A), the
249
pretreatment with nor-BNI, a selective kappa-opioid receptor antagonist (5, 10 or 50
250
µg/paw), reversed the analgesic effect of 4-MAA in a dose-dependent manner (one-
251
way ANOVA followed by Tukey’s test; F5,30 = 46.29; P < 0.05). However, CTOP, a
252
selective mu-opioid receptor antagonist (Fig. 4B; 8, 20, and 32 µg/paw), and
10
253
Naltrindole, a selective delta-opioid receptor antagonist (Fig. 4C; 1, 3, and 9
254
µg/paw), both administered 30 min before 4-MAA injection did not reverse 4-MAA’s
255
analgesic effect (one-way ANOVA followed by Tukey’s test; 4B: F5,30 = 22.97; 4C:
256
F5,30 = 15.05; P > 0.05).
257 258
4 . Discussion
259 260
We have recently demonstrated that dipyrone and its bioactive metabolite 4-MAA
261
have a similar analgesic effect, and both induced activation of L-argenine-NO-KATP
262
pathway (Dos Santos et al., 2014). In the current study, we showed that dipyrone is
263
quickly hydrolyzed to 4-MAA in peripheral tissue, suggesting that dipyrone’s
264
analgesic effect could be mediated by 4-MAA. We also showed that 4-MAA’s
265
analgesic effect is dependent on CB2 and kappa-opioid receptor activation.
266 267
Our results demonstrated that dipyrone or 4-MAA, locally administered in peripheral
268
tissue, inhibit the carrageenan-induced hyperalgesia, which corroborate with
269
previous studies that have demonstrated dipyrone’s analgesic effect in various pain
270
models (Beirith et al., 1998; Dos Santos et al., 2014; Edwards et al., 2010; Rogosch
271
et al., 2012; Vazquez et al., 2005). Carrageenan is an inflammatory agent that
272
induces hyperalgesia by release of endogenous prostaglandins, especially PGE2,
273
which ultimately sensitizes the nociceptors (Lorenzetti and Ferreira, 1985).
274 275
Although dipyrone is metabolized into two bioactive compounds, 4-MAA and 4-AA,
276
this study demonstrated that either dipyrone or 4-MAA prevented the hyperalgesia
277
induced by the inflammatory agent carrageenan, suggesting that the metabolite 4-
11
278
MAA alone is enough to induce effective analgesic effect despite 4-AA.
279
Furthermore, dipyrone was completely hydrolyzed to 4-MAA in the peripheral tissue,
280
confirming our hypotheses that dipyrone’s analgesic effect is mediated by local
281
hydrolysis to 4-MAA (Dos Santos et al., 2014). In fact, some studies have shown
282
that dipyrone’s hydrolysis to 4-MAA is a non-enzymatic reaction that depends on
283
concentration, pH, and temperature (Cohen et al., 1998; Pierre et al., 2007).
284 285
The findings of this study also demonstrated after local dipyrone administration the
286
metabolite 4-MAA can be detected in the peripheral tissue in two forms: protonated
287
[4-MAA+H]+ and sodiated [4-MAA+Na]+. The solution analyzed by HESI before
288
local administration in the peripheral tissue showed a higher sodium concentration
289
(sodiated adduct) when compared with the same solution administrated in the tissue
290
(protonated adduct). This lower concentration of sodium in the tissue and higher of
291
proton is probably because sodium solubilized throughout the tissue creating a
292
lower ionization of this particular ion form. However, independently of the form that
293
the molecule was detected, sodiated or protonated, the results demonstrated that
294
the only product detected was 4-MAA.
295 296
Our results have also shown that 4-MAA’s antihyperalgesic effect depends on CB2
297
cannabinoid receptor activation and kappa-opioid receptors activation. Although
298
CB2 receptor activation can modulate the immune response in peripheral tissue
299
(Lunn et al., 2006), data of this study strongly suggests that 4-MAA decreases the
300
inflammatory hyperalgesia through releasing endogenous opioids followed by
301
kappa-opioid receptor activation. There is robust data showing that CB2 receptor
302
activation induces analgesia by releasing endogenous opioids, which then
12
303
hyperpolarize peripheral nociceptors (Negrete et al., 2011). Therefore, it is plausible
304
to hypothesize that the antihyperalgesic effect of dipyrone in the peripheral tissue is
305
not associated with an anti-inflammatory effect but with the release of endogenous
306
opioids. Nevertheless, because CB2 receptor is probably not expressed in
307
peripheral nociceptors (Freund et al., 2003), the analgesic effect of dipyrone, or
308
more specifically 4-MAA in peripheral tissue may depend on migration of
309
inflammatory cells. These data may explain previous results from our laboratory,
310
where we showed that the analgesic effect of dipyrone or 4-MAA in PGE2-induced
311
hyperalgesia does not depend of CB2 receptor activation (Dos Santos et al., 2014).
312
However, with the carrageenan-induced hyperalgesia model, the analgesic effect of
313
4-MAA was completely prevented by a CB2 receptor antagonist. This difference
314
could be explained by the fact that PGE2 (100 ng) does not induce cell migration to
315
the same degree as carrageenan (Cunha et al., 2008).
316 317
A crosstalk between cannabinoid and opioid systems in pain modulation has been
318
suggested (Machado et al., 2014; Negrete et al., 2011; Guida et al., 2012). Although
319
it is still unclear the mechanism of this interaction, the crosstalk between these two
320
systems has been shown to reduce acute inflammatory or chronic pain (Machado et
321
al., 2014;Guida et al., 2012). Indeed, salvinorin’s analgesic effect depends of CB1
322
and kappa-opioid receptor (KOR), moreover a synthetic KOR agonist is
323
counteracted by AM251, a CB1 antagonist, suggesting a crosstalk between
324
KOR/CB1 receptor (Guida et al., 2012). Also, significant data has been published
325
demonstrating a functional relationship between CB2/ KOR, in fact CB2 receptor
326
activation has been shown to induce endogenous opioid releasing triggering KOR
327
receptor activation (Machado et al., 2014; Negrete et al., 2011).
13
328 329 330
The endocannabinoid system is composed of endogenous ligands anandamide
331
(AEA, arachidonoylethanolamide) and 2-arachidonoylglycerol (2-AG) and
332
cannabinoid CB1 and CB2 receptors, expressed in central and peripheral nervous
333
systems. Whereas data of this study demonstrated that the antihyperalgesic effect
334
of 4-MAA is reversed by AM630, a selective CB2 receptor antagonist, this study
335
does not demonstrate the mechanism underlying CB2 receptor activation by 4-MAA.
336
Indeed, recent data has shown that 4-MAA and 4-AA are a precursor of
337
arachidonoyl amides that induces stimulation of CB1 and CB2 receptors (Rogosch et
338
al., 2012). Data from our laboratory broaden this perspective, showing that the
339
analgesic effect of 4-MAA depends on CB2 receptor activation. Moreover, 4-MAA
340
analgesic effect does not involve CB1 receptor activation in carrageenan-induced
341
pain corroborating our previous, which used prostaglandin as model of pain (Dos
342
Santos et al., 2014). Although carrageenan and prostaglandin induce pain in
343
different ways, carrageenan overlap PGE2 as a final inflammatory mediator that
344
sensitize the nociceptors (Lorenzetti and Ferreira, 1985). These data reinforce that
345
4-MAA induced analgesia independently of the CB1 receptor. On the other hand, 4-
346
AA’s analgesic effect is mediated by CB1 receptor when its antihyperalgesic effect is
347
prevented by the administration of AM251 a selective cannabinoid CB1 receptor
348
antagonist (Dos Santos et al., 2014).
349 350
Because this study demonstrated the involvement of kappa-opioid receptor, but not
351
mu or delta opioid receptors, it is possible that the endogenous opioid released by
352
CB2 receptor activation after 4-MAA is dynorphin. Previous data from our laboratory
14
353
demonstrated that the administration of supernatant of primary culture of
354
keratinocytes or fibroblasts blocks the hyperalgesia induced by carrageenan in rat
355
hind paw, which is prevented by mu, but not by delta or kappa opioid receptor
356
antagonists. These data may rule out the involvement of these cells in the
357
antihyperalgesic effect of 4-MAA. On the other hand, it has been demonstrated that
358
adoptive transfer of M2, but not M0 or M1 macrophage, alleviates the inflammatory
359
hyperalgesia by release of endogenous opioids, including dynorphin (Pannell et al.,
360
2016). Future studies in our laboratory will address the involvement of macrophages
361
in the antihyperalgesic effect of dipyrone and its metabolites. In agreeing with our
362
results, it was recently demonstrated that crotalphine, a non-opioid peptide, induces
363
antihyperalgesic effect dependent on the peripheral CB2 cannabinoid receptor
364
activation and the subsequent release of dynorphin (Machado et al., 2014).
365 366
In conclusion, data of this study suggest a new mechanism of action for dipyrone as
367
well as 4-MAA, which involves an interaction between peripheral CB2 and opioid
368
systems. This interaction represents a new strategy to treat inflammatory pain
369
without inducing central nervous system-mediated undesirable effects and point to
370
4-MAA as a bioactive metabolite and new therapeutic for the treatment of
371
inflammatory pain with minimal side-effects.
372 373 374 375
Acknowledgements This work was funded by CAPES (Coordination of Improvement of Higher Education Personnel).
376 377
Conflict of interest
15
378
The authors declare no conflict of interest.
379 380
Fig.s Legends
381
Fig. 1. Local administration of dipyrone or 4-methylaminoantipyrine (4-MAA) in
382
peripheral tissue reduces the carrageenan-induced hyperalgesia. Dipyrone (Dipy) or
383
4-MAA administered in the hindpaw 2.5 h after carrageenan inhibited, in a dose-
384
dependent manner, the mechanical hyperalgesia evaluated 30 min later
385
(respectively Fig. 1A, 1B). Dipyrone or 4-MAA (160 µg/paw, but not 320 µg/paw),
386
administered in contra-lateral paw (Ct) did not change the mechanical withdrawal
387
threshold, ruling out its systemic effect. The symbol “*” means different from control
388
group (0.9% NaCl administration; 50 µL), “#” means different from carrageenan
389
group (one-way ANOVA followed by Tukey’s test; P < 0.05).
390 391
Fig. 2. Dipyrone is hydrolysate to 4-MAA in the hindpaw. (2A) In Mass spectrum the
392
presence of two peaks can be noticed, the first at 218 m/z (1.65 ppm) and the
393
second 240 m/z (2.16 ppm). These peaks were assigned to the molecular peak of
394
4-MAA [4-MAA+H]+ and the adduct of the 4-MAA with Na, [4-MAA+Na]+
395
respectively. The dipyrone administration in the subcutaneous tissue of hindpaw
396
distributed in [4- MAA+H]+ and [4-MAA+Na]+ (Fig. 2B and 2C, respectively) in a
397
similar tissue area when analyzed by DESI-MSI. No signal of dipyrone (MW: 333
398
g/mol) was found in the mass spectrum.
399 400
Fig. 3. Antihyperalgesic effect of 4-methylaminoantipyrine (4-MAA) is mediated by
401
CB2 and opioid receptor activation. Local administration of carrageenan in rat´s
402
hindpaw induced mechanical hyperalgesia 3 h after, which is decreased with 4-MAA
16
403
administered 2.5 h after carrageenan. This antihyperalgesic effect was reversed by
404
Naloxone (3A 2, 10 and 20 µg/paw) or AM630 (3B 50 and 150 µg/paw) but not by
405
AM251 (3C 80 and 240 µg/paw). The symbol “*” means different from control group
406
(0.9% NaCl; 50 µL), “#” means different from carrageenan group and “&” means
407
different from carrageenan plus 4-MAA (one-way ANOVA followed by Tukey’s test;
408
P < 0.05). The vehicle’s administration of 4-MAA or AM630 (propylene glycol+0.1%
409
DMSO) does not change the mechanical withdraw threshold by itself.
410 411
Fig. 4. Antihyperalgesic effect of 4-methylaminoantipyrine (4-MAA) is mediated by
412
kappa-opioid receptor activation. Local administration of carrageenan in rat´s
413
hindpaw induced mechanical hyperalgesia 3 h after, which is decreased with 4-MAA
414
administered 2.5 h after carrageenan. This antihyperalgesic effect was reversed by
415
nor-BNI, a selective Kappa-opioid receptor antagonist (4A 5, 10 and 50 µg/paw) but
416
not CTOP, a selective Mu-opioid receptor antagonist (4B 8, 20 and 32 µg/paw) or
417
Naltrindole, a selective delta-opioid receptor antagonist (4C 1, 3 and 9 µg/paw). The
418
symbol “*” means different from control group (0.9% NaCl; 50 µL), “#” means
419
different from carrageenan group (one-way ANOVA followed by Tukey’s test; P <
420
0.05). The administration of vehicle does not change the mechanical withdraw
421
threshold by itself.
422 423
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