Chemico-Biological Interactions 311 (2019) 108790

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Modulation of COX-2, INF-ɣ, glutamatergic and opioid systems contributes T

to antinociceptive, anti-inflammatory and anti-hyperalgesic effects of bis(3-
amino-2-pyridine) diselenide
Angélica S. Reisa, Ane G. Vogta, Mikaela P. Pinza, Guilherme T. Vossa, Caren A.R. da Fonsecaa,
Jaini J. Paltiana, Thiago J. Peglowb, Rodrigo A. Vaucherc, Joanna V.Z. Echeniqued,
Mauro P. Soaresd, Ricardo F. Schumacherb, Gelson Perinb, Cristiane Luchesea,**,
Ethel A. Wilhelma,*
a
Laboratório de Pesquisa em Farmacologia Bioquímica (LaFarBio)- Grupo de Pesquisa em Neurobiotecnologia, CCQFA, Universidade Federal de Pelotas (UFPel), Pelotas,
RS, Brazil
b
Laboratório de Síntese Orgânica Limpa (LASOL), CCQFA, Universidade Federal de Pelotas (UFPel), Pelotas, RS, Brazil
c
Laboratório de Pesquisa em Bioquímica e Biologia Molecular de Micro-organismos (LaPeBBioM), Universidade Federal de Pelotas (UFPel), Pelotas, RS, Brazil
d
Laboratório Regional de Diagnóstico, Faculdade de Veterinária, Universidade Federal de Pelotas(UFPel), Pelotas, RS, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Preclinical assays play a key role in research in research on the neurobiology of pain and the development of
Nociception novel analgesics. Drugs available for the treatment of inflammatory pain are not fully effective and show adverse
Analgesic effects. Thus, we investigated the antinociceptive, anti-inflammatory and anti-hyperalgesic effects of bis(3-
Anti-edematogenic amino-2-pyridine) diselenide (BAPD), a new analgesic drug prototype. BAPD effects were investigated using
Allodynia
nociception models induced by chemical (glutamate), immunologic (Freund's Complete Adjuvant - CFA) and
Selenium
thermal stimuli in Swiss mice. Mice were orally (p.o.) treated with BAPD (0.1–50 mg/kg) 30 min prior to the
Pyridine
glutamate and hot-plate tests and a time–course (0.5 up to 8 h) of the antinociceptive effect of BAPD (50 mg/kg,
p. o.) was evaluated in a CFA model. In the CFA model, BAPD effects on cyclooxygenase-2 (COX-2), tumor
necrosis factor (TNFα) and interferon-γ (INF-γ) expression, myeloperoxidase (MPO) activity, oxidative (2,2′-
Azino-bis-3-ethylbenzothiazoline 6-sulfonic acid and 2,2-diphe- nyl-1-picrylhydrazyl levels) and histological
parameters were evaluated. The safety of the compound (50 and 300 mg/kg, p. o.) was verified for 72 h. BAPD
reduced the licking time induced by glutamate and caused an increase in latency response to thermal stimulus.
Naloxone reversed the antinociceptive effect of BAPD. Paw edema formation induced by glutamate or CFA
injection was reduced by BAPD. Mechanical hyperalgesia induced by CFA was attenuated by BAPD. BAPD did
not protect against the increase in MPO activity and decrease of the 2,2′-Azino-bis-3-ethylbenzothiazoline 6-
sulfonic acid and 2,2-diphe- nyl-1-picrylhydrazyl levels induced by CFA. BAPD protected against histological
alterations and reduction on the levels of gene expression COX-2 and INF-γ in the paw of mice exposed to CFA.
BAPD was safe at the doses and time evaluated. BAPD exerts acute antinociceptive, anti-inflammatory and anti-
hyperalgesic actions, suggesting that it may represent an alternative in the future development of new ther-
apeutic strategies.

1. Introduction emotional experience associated with actual or potential tissue damage.

In fact, it represents an essential adaptive response to protect the in-
According to epidemiological studies, the worldwide prevalence of tegrity of the organism. However, pain can also be generated by ma-
pain is approximately 30% [1,2]. The International Association for the ladaptive responses of the organism, affecting daily activities and
Study of Pain (IASP) defines pain as an unpleasant sensory and quality of life [3].

*
Corresponding author. Programa de Pós-graduação em Bioquímica e Bioprospecção (PPGBBio), Centro de Ciências Químicas, Farmacêuticas e de Alimentos,
Universidade Federal de Pelotas (UFPel), Campus Capão do Leão, Pelotas, CEP 96010-900, RS, Brazil.
**
Corresponding author.
E-mail addresses: cristiane_luchese@yahoo.com.br (C. Luchese), ethelwilhelm@yahoo.com.br (E.A. Wilhelm).

https://doi.org/10.1016/j.cbi.2019.108790
Received 3 May 2019; Received in revised form 23 July 2019; Accepted 7 August 2019
Available online 07 August 2019
0009-2797/ © 2019 Elsevier B.V. All rights reserved.
A.S. Reis, et al.
Several drugs are available to treat pain and inflammation [4,5].
However, major adverse effects limit the use of these drugs [6]. In this
context, the development of new therapeutic agents for the treatment
pain and inflammation is of great interest. In this sense, our research
group has dedicated itself to studying the synthesis and pharmacolo-
gical properties of new compounds with anti-inflammatory and an-
algesic activities [7–9]. Our strategy is based on the design of new
compounds objectifying structural improvement.
Some authors have suggested that the chemical structure of orga-
nochalcogens plays an important role in establishing their biological
applications [10–12]. Although research groups have studied the
pharmacological properties of organochalcogens [13,14], the study of
pyridin derivatives containing selenium has been little explored.
Indeed, a growing body of evidence demonstrates that pyridine
compounds have attracted much attention in the field of drug devel-
opment [15–18]. It should be considered that several studies have ad-
dressed the potential antinociceptive and anti-inflammatory effects of
pyridine derivatives [19–21]. Similarly, organoselenium compounds
exhibit several pharmacological properties, such as antioxidant, anti-
allodynic, anti-hyperalgesic, antinociceptive and anti-inflammatory
actions [7,22–26]. It is important to highlight the advantages of re-
searching organoselenium compounds. Among others, antioxidant ac-
tion is an important activity because oxidative stress is critical to the
etiology of many diseases. Therefore, the use of compounds with a
potent antioxidant effect, such as organoselenium compounds, has re-
ceived attention from researchers, because they are promising multi-
target therapeutic agents in the animal model [7,10,13,22]. A literature
survey indicates that only few publications have mentioned the in-
corporation of a selenium atom in the pyridine nucleus. Consequently,
synthesis and biological screening of selenopyridine derivatives may be
considered a relevant research area. Among the selenopyridine deri-
vatives, bis(3-amino-2-pyridine) diselenide (BAPD), a compound ob-
tained by simple and environmentally benign protocols, has been stu-
died by our research group and results obtained after preliminary
biological assays show that BAPD has an important potential to act
against oxidative stress and as an inhibitor of acetylcholinesterase ac-
tivity [27].
In this context, considering that (i) the combination of pyridines and
selenium is scarce, and (ii) the designed synthesis of these compounds
has a great potential for the creation of new molecules with biological
applications, we aimed to evaluate the antinociceptive, anti-in-
flammatory and anti-hyperalgesic effects of a new pyridine derivative
containing selenium, BAPD (Fig. 1), in mice. The potential acute toxi-
city and mechanisms involved in the pharmacological actions of BAPD
were also investigated.
2. Materials and methods
2.1. Drugs
BAPD was prepared and characterized in our laboratory and ana-
lysis of the 1H NMR and 13C NMR spectra showed analytical and
spectroscopic data in full agreement with its assigned structure [27].
The chemical purity of BAPD (99.9%) was determined by GC/MS.
BAPD was dissolved in canola oil, while all other drugs were dis-
solved in a saline solution (0.9%) and were of analytical grade and
obtained from standard commercial suppliers. (+)-MK-801 hydrogen
Fig. 1. Chemical structure of bis(3-amino-2-pyridine) diselenide (BAPD).
2
Chemico-Biological Interactions 311 (2019) 108790
maleate (MK-801) was purchased from Sigma (St. Louis, MO, USA).
Mice received oral treatment (p.o.) by intragastric gavage at a constant
volume of 10 ml/kg body weight.
2.2. Animals
All experiments were performed in accordance with the guidelines
of the Committee on Care and Use of Experimental Animal Resources of
the Federal University of Pelotas, Brazil, and were approved by the
same Committee (CEEA 1289–2016) and in accordance with the U.K.
Animals (Scientific Procedures) Act, 1986 and associated guidelines.
The experiments were carried out using male adult Swiss mice
(25–30 g). Animals were maintained at 22 ± 2 °C with free access to
water and food, under a 12:12 h light/dark cycle (with lights on at 6:00
a.m.). The mice were acclimatized to the behavior room for at least 1 h
before the test. Every effort was made to minimize the number of ani-
mals used and their discomfort. The size of the group used for each
experiment was based on studies that used protocols similar to those
proposed here. The number of animals and intensities of noxious sti-
muli used were the minimum needed to demonstrate the consistent
effects of the treatments. The allocation was concealed using a rando-
mization procedure (http://www.randomizer.org/). Behavioral eva-
luations were performed blindly on drug administration. Each experi-
ment was repeated two to three times (using two or three animals for
each repetition) and experiments were carried out between 08:00 and
17:00 h.
2.3. Experimental protocol
2.3.1. Glutamate-induced nociception
The glutamate test was chosen for this study because it is largely
used to screen new drugs with an antinociceptive effect. The procedure
used was similar to that described previously [28]. Mice were treated
with BAPD (0.1–50 mg/kg, p. o.) or vehicle (canola oil, p. o., 10 ml/kg)
30 min before intraplantar (i.pl.) injection of glutamate (20 μmol/paw,
20 μl) into the right hind paw and saline solution (0.9%, w/v; 20 μl/
paw) into the left paw. Mice were observed individually for 15 min
following glutamate injection and the amount of time spent licking the
injected paw was recorded with a chronometer and it was considered as
a nociceptive behavior. The BAPD pretreatment time was defined in
accordance with previous studies that observed the pharmacological
effects of organoselenium compounds 30 min after pretreatment
[7–9,29–33].
After the observation period, the mice were killed, and the edema
was measured. Paws were removed and weighed. The paw edema was
measured by comparing the difference between the weight of the glu-
tamate-injected paw and the weight of the contralateral paw (saline-
treated paw).
2.3.2. Evaluation of the involvement of the N-methyl-d-aspartate (NMDA)
receptor in the antinociceptive effect of BAPD
The possible contribution of the glutamatergic system to the anti-
nociceptive action of BAPD was investigated using a glutamate-induced
nociception test [28]. NMDA receptor involvement in the anti-
nociceptive action of BAPD was evaluated using MK-801 (0.02 mg/kg
intraperitoneal, i. p.), an uncompetitive antagonist of the NMDA re-
ceptor. Fifteen min after the MK-801 or vehicle administration (10 ml/
kg of body weight, i. p.), the animals received BAPD (50 mg/kg, p. o.)
and after 30 min, the glutamate injection. The pretreatment times,
doses and route of administration were selected in accordance with a
previous study [33].
2.3.3. Hot-plate test
The hot-plate test is used as a model of acute non-inflammatory
nociception to investigate the central effect of analgesic drugs. The
procedure was carried out as described by Woolfre and MacDonald
A.S. Reis, et al.
(1944) [34]. It is a behavioral model of nociception where behaviors
such as jumping and hind paw-licking are elicited following a noxious
thermal stimulus. For this, animals were placed in a glass box on a
heated metal plate maintained at 55 ± 1 °C. The latency of nociceptive
responses such as licking or shaking one of the paws or jumping was
recorded as the reaction. In order to avoid damage to the paws of an-
imals, time standing on the plate was limited to 45 s. The change in
thermal withdrawal latencies was recorded before treatment and at
30 min after treatment with BAPD (0.1–50 mg/kg, p. o.) or vehicle. The
BAPD pretreatment time was defined in accordance with previous
studies that observed the pharmacological effects of organoselenium
compounds at 30 min after pretreatment [7–9,29–33]. The delta of la-
tency (Δt) was calculated for each animal: Δt (s) = post-treatment la-
tency – pretreatment latency.
2.3.4. Evaluation of the involvement of the opioid system in the
antinociceptive effect of BAPD
Opioids have been reported to cause analgesia in rodents in the hot-
plate test [35,36]. Thus, the possible participation of the opioid system
in the BAPD antinociceptive effect was investigated in the hot-plate test
as described above. To this end, the animals were pretreated with na-
loxone (5 mg/kg, subcutaneous, s. c.) or saline (0.9% NaCl) 15 min
before the administration of vehicle (canola oil, p. o., 10 ml/kg), mor-
phine (2.5 mg/kg, s. c.) or BAPD (50 mg/kg, p. o.). The pretreatment
times, doses and route of administration were selected in accordance
with a previous study [23].
2.3.5. Open field test (OFT)
OFT evaluates the general locomotor and exploratory behaviors of
mice. The open-field was made of plywood and surrounded by 30 cm -
high walls. The floor of the open-field, 45 cm long and 45 cm wide, was
divided by masking tape markers into 9 squares (3 rows of 3). Thirty
min after the treatments, each animal was placed at the center of the
open field and observed for 4 min to record the locomotor (number of
segments crossed with the four paws) and exploratory (number of
rearings on the hind limbs) activities [37]. The arena was cleaned with
70% ethanol after each session and individual mice were tested only
once.
2.3.6. Mechanical withdrawal threshold and paw edema induced by
complete Freund's adjuvant (CFA)
Based on the results obtained in the nociception models, to extend
our knowledge of the pharmacological properties of BAPD, it was tested
in an inflammatory pain model. The effect of BAPD was investigated in
an immunologic reaction induced by i. pl. injection of CFA. The me-
chanical hyperalgesia was measured as described by Bortolanza et al.
(2002) [38]. The frequency of withdrawal was determined before
(baseline) CFA injection, in order to obtain data purely derived from the
treatments in CFA allodynia. In this experiment, the time-course of the
antinociceptive effect of BAPD was evaluated at the dose of 50 mg/kg
(p.o.). Mice received the i. pl. injection of CFA (1 mg/ml of heat killed
Mycobacterium tuberculosis in 85% paraffin oil and 15% mannide
monoleate, 20 μl) into the right hind paw and saline solution (0.9%, w/
v; 20 μl/paw) into the left paw. The inflammatory response was verified
24 h after CFA injection into the i. pl. surface of the right hind paw in
mice. Therefore, mice that presented hyperalgesia received vehicle
(canola oil, p. o., 10 ml/kg) or BAPD (50 mg/kg, p. o.) and the with-
drawal response frequency in von frey filament (VHF) was recorded
0.5, 2, 4, 6 and 8 h after treatments. Mechanical hyperalgesia in mice
was measured by using a calibrated nylon VHF of 1 g.
After the test, the animals were killed, and both paws were cut at
the ankle joint and immediately weighed on an analytical balance. The
paw edema was measured by comparing the difference between the
weight of the CFA-treated paw and the weight of the contralateral paw
(vehicle - treated paw).
3
Chemico-Biological Interactions 311 (2019) 108790
2.4. Ex vivo assays, qT-PCR and histological evaluation
In order to improve the understanding of the mechanisms involved
in the pharmacological properties of BAPD, the CFA model was used
according to the protocol described above. Four hours after the BAPD
treatment, the animals were anesthetized and then killed to collect
samples from both paws that were cut off at the ankle joint and used for
histological evaluation as well as to determine the myeloperoxidase
(MPO) activity, 2,2′-azino-bis-3-ethylbenzothiazoline 6-sulfonic acid
(ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals levels. For
the RNA extraction and expression of cyclooxygenase-2 (COX-2), tumor
necrosis factor (TNFα) and interferon-γ (INF-γ), the samples were im-
mediately processed and adequately stored (−80 °C) until the evalua-
tion of the expression.
2.4.1. MPO assay
MPO activity was assayed according to the method previously de-
scribed [39] with some modifications. The paws were homogenized in
potassium phosphate buffer (20 mmol/l, pH 7.4) containing ethylene-
diaminetetraacetic acid (0.1 mmol/l). After the homogenization, sam-
ples were centrifuged at 900×g at 4 °C for 10 min to yield a low-speed
supernatant fraction (S1). Then, S1 fraction was centrifuged again at
20,000×g at 4 °C for 15 min to yield a final pellet (P2) that was re-
suspended in medium containing potassium phosphate buffer
(50 mmol/l, pH 6.0) and hexadecyltrimethyl ammonium bromide
(0.5%). Samples were finally frozen and thawed three times for the
later enzymatic assay. For the MPO activity measurement, an aliquot of
resuspended P2 (100 μl) was added to a medium containing suspension
medium and N,N,N′,N'-tetramethylbenzidine (1.5 mmol/l). The kinetic
analysis of MPO was started after the addition of H2O2 (0.01%), and
color reaction was measured at 655 nm at 37 °C. Results were expressed
as optic density (OD)/mg protein/min.
2.4.2. DPPH and ABTS radicals scavenging activity assay
Paw tissue was collected and immediately homogenized with 500 μl
of potassium chloride (1.15%), and centrifuged (for 10 min, at 200×g,
at 4 °C). The ability of the sample to resist oxidative damage was de-
termined as free radical scavenging ability by ABTS and DPPH assays
[40,41]. For an ABTS assay, ABTS solution was diluted with phosphate
buffer saline pH 7.4 (PBS) to an absorbance of 730 nm. Then, 1.0 ml of
diluted ABTS solution was mixed with 20 μl of S1. After 6 min, the
absorbance was measured at 730 nm. For DPPH assay, DPPH solution
was diluted with ethanol to an absorbance of 517 nm. Then, 1.0 ml of
diluted DPPH solution was mixed with 20 μl of S1. After 6 min, the
absorbance was measured at 517 nm. The results were equated against
an ascorbic acid standard curve. The results were expressed as
equivalents of ascorbic acid per gram of tissue weight in both assays.
2.4.3. RNA extraction and expression of COX-2, TNFα and INF-γ by real-
time PCR
The total RNA of paw tissue was directly isolated after the com-
pletion of treatment using TRIZOL® (Invitrogen, Carlsbad, CA) ac-
cording to the manufacturer's instructions. Total RNA was treated with
RNase-free DNase (Invitrogen, Carlsbad, CA) and its quality was as-
sessed by running samples on a 1% formaldehyde agarose gel. RNA was
quantified spectrophotometrically. The primers used for the real-time
PCRs were synthesized by Invitrogen (São Paulo, Brazil). The real-time
PCR amplification reaction was carried out using SYBR® Green One-
Step qRT-PCR with Rox (Invitrogen, Carlsbad, CA), performed ac-
cording to the manufacturer's protocol. cDNA was synthesized from
0.5 μg of total RNA, using the specific forward and reverse primers
(20 μM) for each gene. PCR reactions were run in a 7500 Real time Fast
thermocycler (Applied Biosystems), under the following conditions:
50 °C for 15 min, 95 °C for 52 min, followed by 40 cycles at 95 °C for
15 s and 60 °C for 30 s, after the dissociation curve was performed at
95 °C for 5 min with a final step of 4 °C. The assay was accomplished for
A.S. Reis, et al.
each gene and included cDNA of the samples treated and control
without template.
Results were obtained as CT (threshold cycle) values. The software
determines a threshold line at the base of the baseline fluorescent
signal, and the data point that meets the threshold is given as CT, which
is inversely proportional to the starting template copy number. The
differences in CT values between the control group, treated group and
endogenous control β-actin gene for each reaction (ΔCT) were analyzed
using the 2−ΔΔCT method. The relative expression genes after incuba-
tion were determined by dividing expression units of the treated group
by the control group. All measurements were performed in duplicate in
two independent experiments. The results were expressed as relative
concentration calculated as described by Giongo et al. (2017) [42].
2.4.4. Protein determination
The protein concentration was measured by the method of Bradford
(1976) [43], using bovine serum albumin as the standard.
2.4.5. Histological examination
Paws were collected and fixed by immersion in 10% buffered for-
malin solution for 24 h. Subsequently, the hind paws were decalcified in
8% hydrochloric acid and formic acid solution at a ratio of 1:1 over a
period of approximately 5 days. The samples were crosscut and routi-
nely processed, embedded in paraffin, cut into of 3–4 μm sections,
stained with hematoxylin-eosin (HE) and examined under an optical
microscope by two independent pathologists.
2.5. Acute toxicity
For evaluation of acute toxicity, mice received a single oral dose of
BAPD (50 and 300 mg/kg) or vehicle. After drug administration, ani-
mals were observed for up to 72 h to determine the lethal potential of
this compound. After this time of exposure, mice were anesthetized for
blood collection by heart puncture in tubes containing heparin. Plasma
was obtained by centrifugation at 900×g for 10 min and used for bio-
chemical assays, which were performed with commercial tests kits.
Plasma aspartate (AST) and alanine aminotransferase (ALT) activities,
used as the biochemical markers for the early acute hepatic damage,
were determined by the colorimetric method [44]. Renal function was
analyzed by determining plasma urea [45]. AST and ALT activities were
expressed as units (U)/l and urea levels were expressed as mg/dl.
Samples of kidney, liver and brain were also collected to determine
non-protein thiols (NPSH) and thiobarbituric acid reactive species
(TBARS) levels and δ-aminolevulinate dehydratase (δ-ALA-D) activity.
Samples were homogenized in 50 mM Tris/HCl pH 7.5 (1:10 for liver/
kidney and 1:5 for brain, w/v) and centrifuged at 900×g for 10 min at
4 °C to yield a S1.
TBARS levels, a measure of lipid peroxidation, were determined as
described by Ohkawa et al. (1976) [46]. An aliquot of S1 was incubated
with 0.8% thiobarbituric acid (TBA), acetic acid buffer pH 3.4 and 8.1%
sodium dodecil sulphate at 95 °C for 2 h. The color reaction was mea-
sured at 532 nm. TBARS levels were expressed as nmol mal-
ondialdehyde (MDA)/mg protein.
NPSH levels were determined as described by Ellman (1968) [47].
An aliquot of S1 was mixed (1:1) with 10% trichloroacetic acid (TCA)
and centrifuged at 900×g for 10 min. After the centrifugation, the
protein pellet was discarded and free –SH groups were determined in
the clear supernatant. An aliquot of S1 was added in 1 M potassium
phosphate buffer, pH 7.4, and 10 mM 5,5-dithiobis (2-nitrobenzoic
acid) (DTNB). The color reaction was measured at 412 nm. NPSH levels
were expressed as μmol NPSH/g tissue.
δ-ALA-D activity was assayed by the method of Sassa (1982) [48].
An aliquot of S1 was pre-incubated for 10 min at 37 °C. The enzymatic
reaction was initiated by adding the substrate (δ-ALA) to a final con-
centration of 2.2 mM in a medium containing 45 mM phosphate buffer,
pH 6.8 and incubated for 3 h (brain) or 1 h (liver/kidney) at 37 °C. The
4
Chemico-Biological Interactions 311 (2019) 108790
incubation was stopped by adding 10% TCA solution with 10 mM
HgCl2. The reaction product porphobilinogen (PBG) was measured at
555 nm using modified Ehrlich's reagent. The values were expressed as
nmol PBG/mg protein/h.
2.6. Statistical analysis
The data and statistical analysis comply with the recommendations
on experimental design and analysis in pharmacology [49]. The raw
data supporting the conclusions of this manuscript will be made
available by the authors, without undue reservation, to any qualified
researcher. The normality of data was evaluated by the D'Agostino and
Pearson omnibus normality test. Statistical analysis was performed
using GraphPad Prism 6.0 software (San Diego, CA, USA). Data were
analyzed by one-way or two-way analysis of variance (ANOVA) fol-
lowed by the Newman–Keuls or Tukey's test when appropriate. Main
effects are presented only when the higher second order interaction was
non-significant. For mechanical withdrawal threshold assays the data
were analyzed using a non-paired t-test followed by the Mann-Whitney
test. Data were expressed as mean ± standard error of the mean
(S.E.M.). Probability values less than 0.05 (P < 0.05) were considered
statistically significant. Post hoc tests were performed only when the F-
value achieved the necessary level of statistical significance (P < 0.05)
and when there was no significant variance in homogeneity [49].
3. Results
3.1. Experimental protocol
3.1.1. Glutamate-induced nociception
The antinociceptive effects exerted by BAPD in the glutamate test
are presented in Table 1. The one-way ANOVA revealed that pretreat-
ment with BAPD reduced the nociception induced by glutamate (F
(4,25) = 78.91, P < 0.0001). The BAPD antinociceptive effect on the
glutamate test was evidenced at the doses of 0.1–50 mg/kg and it was
effective in reducing the licking time induced by glutamate in 100%
(P < 0.0001) at the dose of 50 mg/kg.
Additionally, the pretreatment with BAPD reduced the paw edema
formation induced by glutamate injection in 58% (5 mg/kg;
P < 0.0001), 50% (25 mg/kg; P < 0.0001) and 36% (50 mg/kg;
P < 0.0001) (F (4,25) = 26.28, P < 0.0001) (Table 1).
3.1.2. Evaluation of the involvement of the NMDA receptor in the
antinociceptive effect of BAPD
Fig. 2A llustrates the effect of BAPD administration on glutamate
test after the pretreatment of mice with MK-801. The two-way ANOVA
revealed non significant BAPD x MK-801 (F (1,20) = 0.1458, P > 0.05)
interaction, but significant main effect of BAPD (F (1,20) = 74.01,
P < 0.0001). However, the pretreatment of animals with MK-801 did
Table 1
Effects of BAPD on glutamate and hot plate tests in mice.
Glutamate test Hot-plate test
Groups Licking (s) Paw Edema (mg) Δ Latency (s)
Induced 101.5 ± 9.1 78.7 ± 2.6 −6.0 ± 1.9
BAPD
0.1 mg/kg 74.0 ± 6.3*** 68.9 ± 5.6 5.6 ± 2.3
5 mg/kg 1.0 ± 4.1**** 33.5 ± 3.2**** −0.5 ± 0.9
25 mg/kg 0.0 ± 0.8**** 39.2 ± 4.0**** 17.6 ± 3.6***
50 mg/kg 0.0 ± 0.0**** 49.4 ± 2.3**** 12.7 ± 6.0**
Each column represents the mean ± S.E.M. of 6 mice in each group. BAPD
means bis(3-amino-2-pyridine)diselenide. Asterisks denote significance levels
when compared with the induced group (**) P < 0.01; (***) P < 0.001 and
(****) P < 0.0001 (one-way ANOVA followed by the Newman– Keuls' test).
A.S. Reis, et al. Chemico-Biological Interactions 311 (2019) 108790

Fig. 2. Investigation of the involvement of NMDA receptor and opioid system in

the antinociceptive action of bis(3-amino-2-pyridine) diselenide (BAPD) in
mice. (A) Effect of MK-801 (0.02 mg/kg, i. p.) on the antinociceptive action of
BAPD (50 mg/kg, p. o.) on the licking behavior induced by glutamate in mice;
(B) effect of naloxone (5 mg/kg, s. c.) on the antinociceptive action of BAPD
(50 mg/kg, p. o.) or morphine (2.5 mg/kg, s. c.) on the hot-plate test. Each
column represents the mean ± S.E.M. of 6 mice in each group. (****)
P < 0.0001 and (**) P < 0.01 denotes significance levels when compared with
the control group, (##) P < 0.01 denotes significance levels when compared
with the MK-801 group, (&&) P < 0.01 and (&&&&) P < 0.0001 denotes sig-
nificance levels when compared with the naloxone group (Two-way ANOVA
followed by the Newman– Keuls' test).

not reverse the antinociceptive effect of BAPD (50 mg/kg) in the glu-
tamate test.
3.1.3. Hot-plate test
Effects of BAPD administration in a hot-plate test were demon-
strated in Table 1. The one-way ANOVA revealed that the pretreatment
with BAPD increased response latency to thermal stimulus when com-
pared with the control group (F (4,25) = 7.671, P < 0.001). BAPD
showed significant antinociceptive activity at the doses of 25 and
50 mg/kg, increasing the latency time by 24% (P < 0.001) and 19%
(P < 0.01), respectively.
3.1.4. Evaluation of the involvement of the opioid system in the
antinociceptive effect of BAPD
The two-way ANOVA revealed significant BAPD x naloxone (F
(2,30) = 11.74, P < 0.001) and morphine x naloxone (F (1,20) = 16.83,
P < 0.001) interactions. Pretreatment of animals with naloxone re-
versed the antinociceptive effect of morphine and BAPD on the hot-
plate test (Fig. 2B).
Fig. 3. Effect of bis(3-amino-2-pyridine) diselenide (BAPD) (50 mg/kg, p. o.) on
mechanical hyperalgesia induced by CFA in the ipsilateral paw (A) and in the
contralateral paw (B) of mice in response to 10 applications of 1.0 g VFH. The
animals received vehicle (●) or BAPD (Δ) 24 h after CFA injection. The baseline
(□) was recorded before CFA injection. The measure that follows was 24 h after
CFA-injection (0) and (0.5, 1, 2, 4, 6 and 8 h) subsequent to BAPD treatment.
(C) Effect of BAPD on edema CFA-induced 4 h subsequent to treatment. Each
column represents the mean ± S.E.M. of 7 mice in each group. (*) P < 0.05
and (***) P < 0.001 denote significance levels when compared with the control
group and (#) P < 0.05 denotes significance levels when compared with the
baseline group. Statistical analysis was performed by nonpaired t-test followed
by the Mann Whitney test.
3.1.5. Open field test (OFT)
The possible effects of treatments on locomotor and exploratory
activities of mice were evaluated in the OFT. The data analysis of OFT
revealed that no alteration in the number of crossings (F (4,
25) = 0.3085, P > 0.05) and rearings (F (4, 25) = 2.369, P > 0.05)
were observed after the different treatments of mice (supplementary
material).

5
A.S. Reis, et al.
Table 2
Effects of BAPD (50 mg/kg) on oxidative and inflammatory parameters in the
paw of mice exposed to a VHF test induced by CFA in the 4 h after treatment.
MPO (OD/mg DPPH (equivalents of ABTS (equivalents of
Groups protein/min) ascorbic acid/g of ascorbic acid/g of
tissue) tissue)
Control −0.001 ± 0.0 108.2 ± 5.9 50.2 ± 1.3
Induced 0.128 ± 0.0* 89.4 ± 4.2* 34.5 ± 2.9*
BAPD 0.131 ± 0.0* 89.8 ± 1.9* 40.9 ± 3.4*
Each column represents the mean ± S.E.M. of 6 mice in each group. BAPD
means bis(3-amino-2-pyridine) diselenide. BAPD was evaluated at the dose of
50 mg/kg. Asterisks denote significance levels when compared with the control
group (*) P < 0.05 (one-way ANOVA followed by the Newman– Keuls' test).
3.1.6. Mechanical withdrawal threshold and paw edema induced by CFA
The animals that received BAPD (50 mg/kg, p. o.) demonstrated a
reduction of mechanical hyperalgesia induced by CFA. This hy-
persensitivity reduction started at 0.5 h and remained significant up to
6 h after administration of BAPD (50 mg/kg) (Fig. 3A). The maximal
inhibition observed was 91% at 4 h and the sensitivity of animals was
similar to baseline values (Fig. 3A). No significant difference was ob-
served in the contralateral paw when compared to the basal (Fig. 3B).
Additionally, as verified in Fig. 3C, the treatment with BAPD 50 mg/kg
reduced the paw edema formation (df = 12; t = 4.109, P < 0.01) in-
duced by CFA in 39%.
3.2. Ex vivo assays
3.2.1. MPO assay
The one-way ANOVA revealed that CFA significantly increased the
MPO activity (128 times) in mice paws, when compared with the
control group (F (2,15) = 34.66; P < 0.0001). No protection against the
increase in the MPO activity was elicited after pretreatment with BAPD
(50 mg/kg) in mice (Table 2).
3.2.2. DPPH and ABTS radicals scavenging activity assay
As shown in Table 2, CFA significantly decreased the DPPH (17%)
(F (2,15) = 5.930, P < 0.05) and ABTS (31%) (F (2,15) = 8.14,
P < 0.01) levels in mice paws when compared with the control group
(Table 2). The one-way ANOVA demonstrated that BAPD treatment
(50 mg/kg) did not cause changes in the DPPH and ABTS levels in the
paws with the immunologic reaction induced by CFA (Table 2).
3.2.3. COX-2, TNFα and INF-γ of expression level
As shown in Fig. 4, CFA significantly increased the levels of gene
expression COX-2 (293.2%) (P < 0.0001) and INF-γ (92.1%)
(P < 0.001) in mice paws, when compared with the control group. The
results showed that there was no significant difference in expression of
TNFα after the treatments. The one-way ANOVA demonstrated that
BAPD (50 mg/kg, p. o.) reduced the levels of gene expression COX-2
(P < 0.0001) and INF-γ (P < 0.05) in mice paws, when compared
6
Chemico-Biological Interactions 311 (2019) 108790
with the induced group (Fig. 4).
3.2.4. Histological examination
Briefly, results demonstrated that the mice of the control group had
preserved tissues and no inflammatory process. Meanwhile, the mice of
the induced group (CFA) showed subcutaneous tissue with marked
expansion, edema, prominent lymphatic vessels dilatation and severe
eosinophilic inflammatory infiltrate. Additionally, the CFA caused ex-
tensive damage in the articular region of the mice paw, such as thick-
ened synovial membrane, epithelial cell necrosis, hemorrhage, eosino-
philic inflammatory infiltrate and marked lymphatic vessels dilatation.
However, mice treated with BAPD demonstrated a partial reversal of
the damage caused by the CFA, with reduction of the edema and in-
flammatory process.
As shown in Fig. 5, mice exposed to CFA (Fig. 5B, E, H) presented a
metatarsal-phalangeal region that was enlarged twice the control
group, (Fig. 5A, D, G). CFA induced the flattening of cutaneous folds
due to severe edema and eosinophilic inflammatory infiltrate (Fig. 5B).
The intra-articular space was expanded by severely diffuse eosinophilic
inflammatory infiltrate and the articular surfaces were dissociated.
Cellular debris, edema and hemorrhage were observed in the articular
space. The synovial membrane was thickened by severe eosinophilic
inflammatory infiltrate admixed with chondrocyte necrosis, hemor-
rhage and lymphatic vessel dilatation (Fig. 5E). The interosseous con-
nective and subcutaneous tissues were expanded due to edema, fibrin
and severe eosinophilic inflammatory infiltrate. Additionally, there was
prominent lymphatic vessels dilation and eosinophilic perivasculitis
(Fig. 5B).
BAPD pretreatment reduced the severe articulation and metatarsal-
phalangeal region enlargement, when compared with a CFA group. In
this group, distinct cutaneous folds were present (Fig. 5C). The inter-
osseous connective and subcutaneous tissue presented less severe his-
tological alterations. Mild lymphatic vessel dilatation, moderate eosi-
nophilic inflammatory infiltrate, edema and in some areas calcification
secondary to necrosis were observed (Fig. 5I). The synovial membrane
showed coating epithelial cell loss with less severe congestion and mild
eosinophilic inflammatory infiltrate. There were remaining cellular
debris, hemorrhage and fibrin within the intra-articular space (Fig. 5F).
3.3. Acute toxicity
A single oral administration of BAPD (50 and 300 mg/kg) did not
cause the death of exposed mice. As demonstrated in Table 3, the ac-
tivities of ALT (F (2, 6) = 0.8086, P > 0.05), AST (F (2, 6) = 2.475,
P > 0.05) and the levels of urea remained (F (2, 6) = 0.2183,
P > 0.05) unaltered after BAPD treatment, when compared to those of
mice in the control group.
In addition, effects of a single oral administration of BAPD (50 and
300 mg/kg) on oxidative stress parameters in the brain, kidney and
liver of mice are shown in Table 4. As revealed by statistical analysis,
TBARS (F (2, 6) = 1.738, P > 0.05 for brain; F (2, 6) = 1.647, P > 0.05
Fig. 4. Effect of bis(3-amino-2-pyridine) dis-
elenide (BAPD) (50 mg/kg, p. o.) on levels of ex-
pression of COX-2, TNF-α and INF-γ related genes
in the paw tissue of mice exposed to CFA. The
relative expression ratios are the average values
from three replicate cultures. COX-2, TNF-α and
INF-γ expression levels were normalized using the
geometric mean of β-actin gene. (*) P < 0.05,
(***) P < 0.001 and (****) P < 0.0001 denote
significance levels when compared with the con-
trol group and (#) P < 0.05 and (####)
P < 0.0001 denote significance levels when
compared with the induced group.
A.S. Reis, et al. Chemico-Biological Interactions 311 (2019) 108790

Fig. 5. Photomicrograph of the morphological

alterations of mice paws: (A) Control: Tarsal re-
gion. HE. 40X. (B) CFA (induced): Tarsus region.
HE 40X. (C) BAPD + CFA: Tarsus region. HE 40X.
(D) Control: Articular region. HE. 200X. (E) CFA
(induced): Articular region. Thickened synovial
membrane presents epithelial cell necrosis, he-
morrhage, eosinophilic inflammatory infiltrate
and marked lymphatic vessel dilatation (*). The
appearance of the articular surface is rough due to
the irregularity in the membrane surface. In the
articular space, there was cellular debris and he-
morrhage (arrow). HE. 200X. (F) BAPD + CFA:
Articular region. HE. 200X. (G) Control: Tarsus
region. HE. 200X. (H) CFA (induced): Interosseous
connective tissue. Severe eosinophilic in-
flammatory infiltrate admixed with hemorrhage,
edema and fibrin (*). There is marked lymphatic
vessel dilatation (L). HE. 200X. (I) BAPD + CFA:
Interosseous connective tissue. HE. 200X.

Table 3 thermal nociception models, without causing locomotor disturbances

Effect of a single oral administration of BAPD (50 and 300 mg/kg) on bio- and toxicity. Our data indicate that opioid and glutamatergic systems
chemical parameters in mice. are related to the antinociceptive properties of BAPD in mice. In ad-
Groups AST (U/l) ALT (U/l) Urea (mg/dl) dition, CFA injection induced hyperalgesia, allodynia and histological
alterations in the mice paws, and these alterations were reversed by the
Control 77.2 ± 10.4 104.5 ± 27.2 70.2 ± 9.5 BAPD treatment. Additionally, we showed that BAPD reduced the in-
BAPD
flammatory process induced by CFA by reducing levels of gene ex-
50 mg/kg 66.6 ± 9.4 57.3 ± 3.4 61.9 ± 1.6
300 mg/kg 49.7 ± 5.5 90.5 ± 37.7 68.8 ± 13.4 pression COX-2 and INF-γ.
Glutamate and its receptors play a crucial role in perception and
Data are reported as the mean ± S.E.M. of 3 mice in each group. BAPD means integration of nociceptive signals and in their relay to supraspinal
Bis(3-amino-2-pyridine) diselenide. Statistical analysis was performed using centers. Indeed, glutamate is widely distributed throughout virtually all
one-way ANOVA followed by the Newman– Keuls' test. circuits in the entire nervous system and also co-localized with its re-
ceptors in areas of the brain, spinal cord and periphery that are in-
for kidney; F (2, 6) = 0.4617, P > 0.05 for liver) and NPSH (F (2, volved in pain sensation and transmission [50,51]. In this regard, in-
6) = 0.001852, P > 0.05 for brain; F (2, 6) = 0.2459, P > 0.05 for itially, we sought to determine the antinociceptive activity of BAPD
kidney; F (2, 6) = 1.206, P > 0.05 for liver)levels and δ-ALA-D activity using a model of nociception induced by glutamate. As expected, i. pl.
(F (2, 6) = 0.3795, P > 0.05 for brain; F (2, 6) = 0.4230, P > 0.05 for injection of glutamate in the mouse hind paw induced a nociceptive
kidney; F (2, 6) = 1.177, P > 0.05 for liver) remained unaltered after response and edema. Agonists at these receptors or subcutaneous in-
BAPD (50 and 300 mg/kg) treatment, when compared to the control jection of glutamate into the hind paw have been shown to reduce pain
group (Table 4). thresholds [52–55], and conversely antagonists promote analgesia in
peripheral pain [56]. Our results revealed that BAPD inhibited the
4. Discussion nociceptive response and edema caused by glutamate injection, sug-
gesting potential antinociceptive and anti-inflammatory actions pos-
The findings of the present study indicated, for the first time, that sibly by modulation of the glutamatergic system. Importantly, some
BAPD exerts antinociceptive, anti-inflammatory and anti-hyperalgesic researchers have demonstrated that other organoselenium compounds
actions. BAPD treatment reversed the nociception in chemical and

Table 4
Effect of a single oral administration of BAPD (50 and 300 mg/kg) on oxidative parameters in brain, kidney and liver of mice.
NPSH (μmol/g tissue) TBARS (nmol MDA/mg protein) δ- ALA-D (nmol PBG/mg protein/h)

Brain Kidney Liver Brain Kidney Liver Brain Kidney Liver

Control 1.73 ± 0.06 2.29 ± 0.09 6.55 ± 0.19 0.62 ± 0.04 1.70 ± 0.08 2.16 ± 0.16 1.09 ± 0.02 0.79 ± 0.10 8.83 ± 0.29
BAPD
50 mg/kg 1.73 ± 0.01 2.18 ± 0.27 5.57 ± 0.63 0.61 ± 0.02 2.21 ± 0.31 2.02 ± 0.01 1.12 ± 0.04 0.78 ± 0.02 8.32 ± 0.53
300 mg/kg 1.73 ± 0.03 2.36 ± 0.13 6.26 ± 0.43 0.50 ± 0.07 1.69 ± 0.23 2.02 ± 0.12 1.07 ± 0.03 0.70 ± 0.07 8.09 ± 0.44

Date are reported as the mean ± S.E.M. of 3 mice in each group. BAPD means bis(3-amino-2-pyridine) diselenide. Statistical analysis was performed using one-way
ANOVA followed by the Newman– Keuls' test.

7
A.S. Reis, et al.
exhibit potential antinociceptive in the glutamate test [30–33]. The
antinociceptive effect of organoselenium compounds, at doses equal to
or higher than 0.1 mg/kg, in the glutamate test has been reported
[30,31]. Here, BAPD has a similar or higher antinociceptive potency in
the glutamate test when compared to other organoselenium com-
pounds. In line with these results, the involvement of NMDA receptor in
the antinociceptive action of BAPD was investigated using MK-801 (an
uncompetitive antagonist of the NMDA receptor). Data obtained sug-
gested that the modulation of NMDA receptor did not contribute to the
antinociceptive effect of BAPD. However, considering the results in the
glutamate test, we cannot rule out the involvement of other glutama-
tergic receptors in this pharmacological action of BAPD.
Considering that glutamate is released both peripherally and cen-
trally in response to nociceptive stimulation [57,58] and the effects of
BAPD on the glutamate test, we had objectively evaluated the anti-
nociceptive effect of BAPD on thermal stimulus in the hot-plate test.
Acute non-inflammatory nociception is produced in the hot-plate test,
and it is a good model to investigate the central effect of analgesic drugs
with selectivity for opioid-like drugs [59,60]. Supporting the BAPD
antinociceptive action, the current findings revealed that BAPD reduced
the nociceptive behavior of the thermal stimulus, suggesting a central
action of this compound. In line with these results, it was imperative to
understand whether the opioid system is associated with the anti-
nociceptive effect of BAPD. Significantly, the pretreatment of animals
with naloxone, a nonselective opioid receptor antagonist, reversed the
antinociceptive effect caused by oral administration of BAPD, demon-
strating that the opioid system is implicated in this effect. Indeed, these
results are consistent with previous findings that demonstrated the ef-
fect of selol, an organoselenium compound, on the opioid system. Bu-
jalska and collaborators [61] demonstrated that pretreatment with selol
enhanced the analgesic activity of opioids drugs. Evidence for the in-
volvement of μ-opioid and δ-opioid receptors in the antinociceptive
effect caused by oral administration of m-trifluoromethyl-diphenyl
diselenide in mice was provided by Brüning and collaborators [25]. The
results indicate that m-trifluoromethyl-diphenyl diselenide elicited an-
tinociception in different models of pain through mechanisms that seem
to involve an interaction with the central opioid system, more specifi-
cally μ-opioid and δ-opioid receptors. In addition, m-trifluoromethyl-
diphenyl diselenide was effective to abolish the phenotype induced by
repeated forced swim stress, which was accompanied by changes in the
contents of cortical μ- and κ-opioid receptors [62].
Additionally, a further study was performed to extend our knowl-
edge about the anti-inflammatory potential of BAPD. A CFA-induced
inflammatory pain model was used because it effectively mimics some
characteristics of human arthritis, such as the development of me-
chanical allodynia, edema and induction of inflammation [63]. In the
current study, it was demonstrated that a single oral administration of
BAPD produced antinociceptive, anti-hyperalgesic and anti-edemato-
genic actions in CFA-induced painful inflammation in mice. Revealing a
rapid and long action, BAPD reduced the response frequency of VHF
stimulation at 0.5 h and this effect was maintained for up to 6 h with the
maximal inhibition of 91%. The pharmacological actions of other dis-
elenides in CFA-induced painful inflammation have been demonstrated
by other authors [24,26].
In an attempt to identify the mechanisms involved in these effects of
BAPD, the MPO activity, DPPH and ABTS radical scavenging activities
were determined in paws of mice exposed to CFA. Oxidative stress has
been implicated as a mechanism in the induction and maintenance of
pain and its occurrence has been described in several animal models of
pain [60]. Although this hypothesis is strengthened by the fact that
some antioxidant substances present antinociceptive effects [7,64],
here BPAD did not protect against the reduction of DPPH and ABTS
levels induced by CFA. Additionally, concerning the stimulation of MPO
activity found in animals exposed to CFA, our results demonstrated that
BAPD did not exert a protective effect. MPO is a heme peroxidase highly
expressed in the neutrophils and activated microglial cells [65]. Our
8
Chemico-Biological Interactions 311 (2019) 108790
results suggest that BAPD anti-inflammatory, antinociceptive and anti-
hyperalgesic actions are not related to MPO or scavenger activities. Our
results on MPO are according to the data published. Chagas et al. [26]
demonstrated that CFA increased the MPO activity in the paw tissue of
C57BL/6 mice and, the bis(phenylimidazoselenazolyl) diselenide, an
organoselenium compound, did not reverse this alteration.
As a result of CFA-induced tissue damage several chemical media-
tors, such as interleukin, TNFα, prostaglandin, INF-γ, are released from
the injured cells and the infiltrating immune cells to create a peripheral
inflammatory process. In this process, the peripheral terminals of no-
ciceptors express receptors for many of these inflammatory mediators.
This increase in the peripheral sensitivity of nociceptors in inflamed
tissue contributes to inflammatory pain hypersensitivity or hyperalgesia
[66–68]. In this case, tissue damage that involves up-regulation of INF-γ
contributes to inflammation and probably increases the excitability of
nociceptive neurons. Earlier studies demonstrated that INF-γ could in-
duce nitric oxide synthase (iNOS) and activity immune [69,70]. Our
results revealed that BAPD presented a promising anti-inflammatory
effect, since it reduced the paw edema formation induced by CFA and
glutamate. This effect can be due to the potential inhibitory effect of
BAPD on INF-γ, reducing maintenance of the inflammatory process.
Additionally, tissue damage promotes the release of neuro-
transmitters from the central terminals of nociceptors and increases the
production of prostaglandin or pro-inflammatory cytokines in the spinal
cord. This leads to additional excitation and disinhibition of dorsal horn
neurons and causes abnormal responses to sensory signals from the
periphery [71]. In this sense, prostanoids play a pivotal role in in-
flammation and pain. The role of COX in inflammatory pain has been
extensively studied. Here, it is important to highlight that our results
suggest that the single oral administration of BAPD markedly reduced
the levels of COX-2 expression in injured tissue suggesting an anti-in-
flammatory effect of this compound. Indeed, inhibitors of COX, also
known as nonsteroidal anti-inflammatory drugs (NSAIDs), exert anti-
inflammatory action and are mainly used in the treatment of degen-
erative joint disease, rheumatoid diseases, metabolic disorders and
other diseases that are associated with pain and inflammation [72].
This result is in agreement with the study performed by Marcondes Sari
and collaborators [73] that revealed the effectiveness of the organose-
lenium compound p,p'-methoxyl-diphenyl diselenide in an animal
model of chronic pain by restoring the molecular changes in the COX-2
content.
In addition, the present study revealed that BAPD also was able to
reduce the mechanical allodynia in the inflamed paws and the noci-
ceptive behavior from thermal stimulus in mice. This can be due to its
inhibitory potential effect in the expression of INF-γ and COX-2, as well
as to modulating the opioid and glutamatergic pathways. In addition,
these results corroborate those found in the histopathological analysis
that demonstrated a reduction in the inflammatory process since BAPD
reduced lymphatic vessel dilatation and eosinophilic inflammatory in-
filtrate.
Here, we also demonstrated that the single oral administration of
BAPD did not elicit any sign of acute toxicity and did not modify the
locomotor and exploratory activities of animals. BAPD, at doses of 50
and 300 mg/kg, did not cause death or any significant acute tox-
icological effect. Concerning biochemical parameters of hepatic (ALT
and AST activities) and renal (urea) function, BAPD did not alter any of
these parameters in plasma, suggesting that this compound does not
have any potentially toxic effect on these organs. δ-ALA-D activity, the
parameter of potential organoselenium toxicity [22], was not altered in
brain, kidney and liver by treatment with the single oral administration
of BAPD (50 and 300 mg/kg). Additionally, BAPD did not modify
TBARS and NPSH levels.
5. Conclusion
In summary, BAPD induced antinociceptive, anti-hyperalgesic and
A.S. Reis, et al.
anti-inflammatory effects in mice without altering locomotor activity or
inducing toxicity. These effects of BAPD seem to be due to a modulation
of opioid and glutamatergic systems as well as to reduction of the levels
of expression COX-2 and INF-γ. Our results suggest that BAPD may be a
good, more effective and/or potent prototype for the treatment of pain
and inflammation.
Funding information/acknowledgments
We are grateful to UFPel, CNPq (UNIVERSAL 408874/2016-3,
429859/2018-0) and FAPERGS (PqG 17/2551-0001013-2; PRONEM
16/2551-0000240-1 and PRONUPEQ 16/2551- 0000526-5) for fi-
nancial support. C.L., E.A.W. and G.P are recipients of CNPq fellowship.
This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) -
Finance Code 001.
Conflicts of interest
The authors declare that they have no conflict of interest.
Author contributions
A.R, C.L and E.A.W conceived and designed the study. A.V, M.P,
G.V, C·F, J.P, T.P, R.V, J.E, M.S, R.S and G.P carried out the acquisition,
analysis and interpretation of data. A.R, C.L and E.A.W wrote the
manuscript. E.A.W and C.L supervised the study.
Declaration of interests
The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.cbi.2019.108790.
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