H2S donor GYY4137 ameliorates paclitaxel-induced neuropathic pain in mice

Article abstract of DOI:10.1016/j.biopha.2020.110210

Paclitaxel-induced neuropathic pain (PINP) is a dose-limiting side effect that largely affects the patient's quality of life and may limit the use of the drug as a chemotherapeutic agent for treating metastatic breast cancer and other solid tumors. Recently, a putative role for the gaseous mediator hydrogen sulfide (H₂S) in nociception modulation has been suggested. The aim of the present study was to investigate the potential efficacy of the slow release H₂S donor GYY4137 to alleviate and prevent PINP. Female BALB/c mice that were intraperitoneally (i.p.) injected with paclitaxel (2 mg/kg) for 5 consecutive days developed thermal hyperalgesia, cold and mechanical allodynia and had reduced of H₂S, generation in the spinal cord and paw skin. Treatment of mice with established thermal hyperalgesia with GYY4137 or the analgesic positive control drug gabapentin produced antihyperalgesic activities. The antihyperalgesic activity of GYY4137 was antagonized by the ATP sensitive potassium channels (K_ATP channels) blocker glibenclamide. Co-treatment with GYY4137 and paclitaxel prevented the paclitaxel-induced decrease in H₂S, generation as well as the paclitaxel-induced thermal hyperalgesia, cold allodynia and mechanical allodynia. GYY4137 enhanced paclitaxel's anti-proliferative effects against the breast cancer cell line MCF-7. The present results suggest that GYY4137 alleviates paclitaxel-induced thermal hyperalgesia, via K_ATP channels. GYY4137 prevents PINP possibly by blocking the paclitaxel-induced reduction in the generation of H₂S, in the tissues, while enhancing the anti-cancer activity of paclitaxel, and therefore warrants further research as a candidate for prevention of PINP in clinical settings.

Full text of DOI:10.1016/j.biopha.2020.110210

Biomedicine & Pharmacotherapy 127 (2020) 110210
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
H S donor GYY4137 ameliorates paclitaxel-induced neuropathic pain in
2
mice
a,
a
a
b
Bedoor Qabazard *, Willias Masocha , Maitham Khajah , Oludotun Adebayo Phillips
a
Department of Pharmacology and Therapeutics, Faculty of Pharmacy, Kuwait University, Kuwait
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Health Sciences Centre, Kuwait University, Kuwait
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Paclitaxel-induced neuropathic pain (PINP) is a dose-limiting side effect that largely affects the patient’s quality
Hydrogen sulfide
Paclitaxel
Chemotherapy-induced neuropathic pain
of life and may limit the use of the drug as a chemotherapeutic agent for treating metastatic breast cancer and
other solid tumors. Recently, a putative role for the gaseous mediator hydrogen sulfide (H
modulation has been suggested. The aim of the present study was to investigate the potential efficacy of the slow
release H S donor GYY4137 to alleviate and prevent PINP. Female BALB/c mice that were intraperitoneally (i.p.)
injected with paclitaxel (2 mg/kg) for 5 consecutive days developed thermal hyperalgesia, cold and mechanical
allodynia and had reduced of H S, generation in the spinal cord and paw skin. Treatment of mice with estab-
2
S) in nociception
2
2
lished thermal hyperalgesia with GYY4137 or the analgesic positive control drug gabapentin produced anti-
hyperalgesic activities. The antihyperalgesic activity of GYY4137 was antagonized by the ATP sensitive po-
tassium channels (KATP channels) blocker glibenclamide. Co-treatment with GYY4137 and paclitaxel prevented
2
the paclitaxel-induced decrease in H S, generation as well as the paclitaxel-induced thermal hyperalgesia, cold
allodynia and mechanical allodynia. GYY4137 enhanced paclitaxel's anti-proliferative effects against the breast
cancer cell line MCF-7. The present results suggest that GYY4137 alleviates paclitaxel-induced thermal hyper-
algesia, via KATP channels. GYY4137 prevents PINP possibly by blocking the paclitaxel-induced reduction in the
generation of H
2
S, in the tissues, while enhancing the anti-cancer activity of paclitaxel, and therefore warrants
further research as a candidate for prevention of PINP in clinical settings.
1
. Introduction
patients with CINP [3], but currently only duloxetine has moderate
recommendation for the treatment of CINP [4,5]. The gaseous signaling
molecule hydrogen sulfide (H S) has recently been proposed to play a
Platinum analogues (cisplatin and oxaliplatin), taxanes (paclitaxel),
2
vinca alkaloids (vincristine) and proteasome inhibitors (bortezomib)
are the most common antineoplastic drugs successfully employed as
first-line treatment for several solid and blood cancers, including breast,
lung, colorectal, gastric cancers and multiple myeloma. Although these
compounds have different chemical structures and mechanisms of ac-
tion, they all induce the development of chemotherapy-induced neu-
ropathic pain (CINP) as a common side effect. Some of the symptoms of
neuropathic pain include hyperalgesia (an increased response to nor-
mally painful stimuli), allodynia (pain triggered by normally non-
painful stimuli, such as cloth rubbing against the skin) and spontaneous
sensations such as burning, shooting, numbness, spasm and prickling
role as neuromodulator and cytoprotective mediator in several tissues,
including the cardiovascular and nervous system [6–8]. A putative role
for H
inhaled H
induced by chronic constriction injury of the sciatic nerve in mice [9]
and rats [10]. Moreover, H S-releasing analgesic compounds have been
shown to exert greater potency against different forms of pain [11].
Furthermore, H S has been shown to induce mu opioid receptor-de-
pendent analgesia in a rodent model of visceral pain [12]. A recent
study showed a role of H S donors in relieving established CINP pos-
sibly via activation of voltage-gated Kv7potassium channels [13],
however whether there are changes in H S during CINP, H S donors can
prevent the development of CINP or other potassium channels play a
role in the activity of H S donors against CINP remains to be identified.
The aims of this study were therefore to evaluate the whether GYY4137
2
S in nociception modulation has been suggested. For example,
2
S has been shown to prevent the neuropathic pain behavior
2
2
2
[
1,2]. These symptoms can have a serious negative impact on the
2
2
psychosocial well-being and overall quality of life of the patient. Un-
fortunately, there are no clinically proven effective drugs for the pre-
vention of CINP. A variety of therapeutic agents are used to relieve
2
⁎
Corresponding author.
E-mail address: bedoor.qabazard@hsc.edu.kw (B. Qabazard).
https://doi.org/10.1016/j.biopha.2020.110210
Received 16 February 2020; Received in revised form 25 April 2020; Accepted 28 April 2020
0
753-3322/ © 2020 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(
http://creativecommons.org/licenses/BY-NC-ND/4.0/).

B. Qabazard, et al.
Biomedicine & Pharmacotherapy 127 (2020) 110210
can prevent the development of PINP and alleviate established pacli-
taxel-induced thermal hyperalgesia (therapeutic regimen), whether
with normal saline in the same proportion as the paclitaxel solution,
thus, constituted of about 1.7 % Cremophor EL and 1.7 % ethanol in
normal saline. Paclitaxel (2 mg/kg) or its vehicle was administered to
mice intraperitoneally (i.p.), in a volume of 10 mL/kg, once daily for 5
consecutive days. This treatment regimen has been reported to produce
painful neuropathy and thermal hyperalgesia in mice [17–19]
K
ATP channels are involved in the antihyperalgesic effect of GYY4137,
and whether there are changes in H
tissues during PINP.
2
S levels or generation capacity in
2
. Materials and methods
.1. Drugs and chemicals
Paclitaxel was purchased from Tocris Bioscience (Cat. No. 1097;
2
2
.4. Animal groups and treatment regimens
2
.4.1. Acute (therapeutic) regimen
Mice were treated i.p. with GYY4137 (25, 50 and 100 mg/kg), ga-
Bristol, UK), and all other drugs were purchased from Sigma Aldrich, St.
Louis, MO, except GYY4137 which was synthesized in-house, as de-
scribed by Li et al. [15]. Structural characterization by spectrometric
methods was performed by The General Facilities Science (GF-S), Fa-
culty of Science, Kuwait University, Kuwait. Briefly, GYY4137 was
synthesized as follows: Morpholine (40 mmol) in anhydrous di-
chloromethane (DCM, 12 mL) was added dropwise to a solution of
Lawesson’s reagent (8.0 mmol) in anhydrous DCM (12 mL) under ni-
trogen gas at room temperature and the reaction mixture was stirred for
bapentin (10 mg/kg), used as an analgesic positive control as described
previously [20],or vehicle once on day 7 after first administration of
paclitaxel, when the mice had developed thermal hyperalgesia as pre-
viously described [17]. Reaction latencies to the hot plate test were
measured before drug treatment (two baseline values) and at 0.5, 1,
1
.5, 2, and 3 h post drug treatment.
2
.4.2. Chronic (prophylactic) regimen
GYY4137 50 mg/kg was co-administered with paclitaxel daily for 5
days. The mice were assessed for the development of PINP using the hot
and cold plate tests and dynamic plantar aesthesiometer.
2
h. The resulting precipitate was collected by suction filtration and
washed several times with anhydrous DCM and dried to give a pure
white solid product (66 % yield; melting point 156−159 °C). After
synthesis, the purity and structure of the product were verified using
1
2.5. Assessment of thermal nociception
Proton Nuclear Magnetic Resonance Spectrometry ( H NMR) and Mass
1
Spectrometry. GYY4137 was analyzed by H-NMR spectrum in CHCl3-
For thermal hyperalgesia assessment, animals were individually
placed on a hot-plate (Panlab S.L., Barcelona, Spain) with the tem-
perature adjusted to 55 ± 1 °C. The latency to the first sign of paw
licking, vocalization or jump response to avoid the heat was taken as an
index of pain threshold. Reduction in paw licking or jump response time
latency (RTL) indicates hyperalgesia, whereas an increase in RTL after
drug treatment indicates antinociceptive activity of the drug. Reaction
latencies to hot plate test were measured before (baseline latency), at
day 7 after first injection of paclitaxel, and at various time points after
drug treatment. A cut-off period of 20 s was maintained to avoid da-
mage to the paws. For thermal allodynia test, animals were individually
placed on a cold-plate with the temperature adjusted to 4 ± 1 °C. The
latency to the first sign of paw licking was taken as an index of pain
threshold. Reduction in paw licking RTL indicates allodynia, whereas
an increase in RTL after drug treatment indicates antinociceptive ac-
tivity of the drug. A cut-off period of 60 s was maintained to avoid
damage to the paws.
3
d recorded on a Bruker Avance II 600 NMR spectrometer using solvent
peak as a reference signal, and the Mass spectrum was recorded on a
Waters QToF high resolution / Mass Spectrometer (LC–MS/MS High
1
resolution). H-NMR (600 MHz; (CDCl
3
): δ 2.94–2.97 (4H, broad ap-
for morpholine attached to P), 3.19 (4H,
broad t, J =4.9 Hz, morpholinium (CH N), 3.59 (4H, broad t, J =4.3
Hz, O(CH for morpholine attached to P), 3.74 (4H, broad t, J =4.90
Hz, morpholinium (CH O), 3.82 (3H, s, CH O), 5.28 ("1H", s, 0.5
CH Cl ), 6.87–6.89 (2H, m, arylC-H o-to OCH3) and 8.15−8.05 (2H, m,
arylC-H o-to P). 9.19 (2H, broad signal, morpholinium NH
m/z, ES+): Calcd for C15 PS 376.1044; found 377.1100 (M
H).
2 2
parent q, J =6.0 Hz, N(CH )
2 2
)
2 2
)
2
)
2
3
2
2
+
2
). HRMS
+
(
H
25
N
2
O
3
2
+
2.2. Animals and ethics statements
Female BALB/c mice (8–12 weeks old), weighing approximately
0−30 g, supplied by the Animal Resource Center (ARC) at the Health
2
Sciences Center, Kuwait University were used in this study. All animals
were housed under optimal laboratory conditions, maintained on a
natural light and dark cycle and had free access to food and water ad
libitum. Animals were acclimatized to laboratory conditions before any
tests. All experimental procedures were carried out in strict accordance
with the recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health (NIH
Publication number 85−23, Revised 1985) as approved by the Animal
Welfare and Ethics Committees of Kuwait University. Animals were
handled in compliance with ethical guidelines for research in experi-
mental pain with conscious animals [15], and all efforts were made to
minimize suffering. Whenever possible, animal studies are reported in
accordance with ARRIVE guidelines [16].
2
.6. Assessment of mechanical allodynia
Mechanical allodynia in mice was measured using the dynamic
plantar aesthesiometer (Ugo Basile, Italy). Briefly, mice were left to
habituate for 30 min inside plastic enclosures on top of a perforated
platform before starting a microprocessor which was programmed to
automatically lift a metal filament that exerted a linearly increasing
force (0.25 g/s with cut-off time of 20 s) on the hind paw. A stop signal
was automatically attained, either when the animal removed the paw or
at the cut-off force of 5 g. Withdrawal thresholds in response to the
mechanical stimulus were automatically recorded in grams. The hind
paws were tested at least 3 times. The baseline mechanical threshold
was assessed one day before the induction of the neuropathic pain.
2.3. Induction of neuropathic pain by paclitaxel administration
2.7. Involvement of ATP sensitive potassium channel (KATP channel)
Peripheral painful neuropathy was induced in mice by in-
traperitoneal (i.p.) administration of paclitaxel as previously described
17]. Paclitaxel was dissolved in a solution made up of 50 % Cremophor
EL and 50 % absolute ethanol to a concentration of 6 mg/ml (stored at
20 °C, for a maximum of 14 days) and then diluted in normal saline
NaCl 0.9 %) to a final concentration of 0.2 mg/ml just before admin-
istration. The vehicle for paclitaxel was diluted at the time of injection
To explore the possible contribution of KATP channel in the anti-
[
nociceptive effect of GYY4137, mice with paclitaxel-induced neuro-
pathic pain were pre-treated with glibenclamide (an ATP-sensitive K +
channel inhibitor) 15 min before the injection of either vehicle, or
GYY4137 (50 mg/kg, i.p.). The reaction latencies to the hot plate were
recorded at 2 h after injection of GYY4137 or vehicle.
−
(
2

B. Qabazard, et al.
Biomedicine & Pharmacotherapy 127 (2020) 110210
2
.8. Measurement of plasma H
2
S level
computed) where within each row, columns were compared (simple
effects within each row) i.e. compared each cell mean with every other
cell mean on that row. The differences were considered significant
when p < 0.05. The results were expressed as the mean ± standard
error of the mean (S.E.M).
H
2
S levels were determined by zinc trapping spectrophotometric
assay [14]. Blood samples were collected by cardiac puncture under
terminal anesthesia and plasma was separated. The initial reaction
mixture was made by mixing 50 μl plasma with 200 μl of 1 % zinc
acetate. Then, 200 μl of N,N-dimethyl-p-phenylenediamine sulfate (20
mM in 7.2 M HCl) and 200 μl of FeCl
added. After 10-minute incubation, the absorbance of the reaction
mixture was measured at 670 nm, and H S concentration was calcu-
lated against a standard curve of NaHS solution (25−200 μM).
3. Results
3
(30 mM in 1.2 M HCl) were
3.1. GYY4137 alleviates established paclitaxel-induced thermal
hyperalgesia in an ATP sensitive potassium channel (KATP channel)-
dependent manner
2
2
.9. Measurement of H
2
S tissue synthesizing activity
At the baseline day (day 0), there were no significant differences in
the withdrawal latencies to heat-stimuli between the animal groups.
Paclitaxel-treated mice had significant reduction in RTL (thermal hy-
peralgesia) in the hot plate test on day 7 after first drug administration
compared to the baseline (pretreatment) latency (Fig. 1A; p < 0.05).
Therefore, paclitaxel-treated mice developed thermal hyperalgesia on
day 7 after first administration of paclitaxel, in agreement with pre-
vious evidence [17]. The positive analgesic control drug gabapentin (10
mg/kg) produced antihyperalgesic effect starting after 1.5 h of treat-
ment, i.e. significantly increasing RTL to 5.2 ± 0.2 s, as compared to
3.6 ± 0.2 s for paclitaxel + vehicle group (n = 6; p < 0.05, Fig. 1A).
The antihyperalgesic effect of gabapentin was from 1.5 to 3 h, reached a
peak level after 2 h (RTL 6.1 ± 0.2 s vs. 3.6 ± 0.2 s for paclitaxel +
vehicle group; n = 6; p < 0.01, Fig. 1A). Treatment with GYY4137
produced antihyperalgesic effects with all the tested doses but with
variable start and peak times. A 25 mg/kg dose of GYY4137 produced a
significant antihyperalgesic effect from 1.5 to 3 h after treatment, as
evident by the increased RTL to 5.4 ± 0.1 s (n = 8) compared to
3.5 ± 0.2 s for paclitaxel + vehicle group at 1.5 h and 5.5 ± 0.4 (n =
8) compared to 3.7 ± 0.2 s for paclitaxel + vehicle group (n = 6) at 3 h
(p < 0.01; Fig. 1A) and reached a peak at 2 h (RTL 6.8 ± 0.2 s, n = 8
vs. 3.6 ± 0.2, n = 6 paclitaxel + vehicle-treated mice; p < 0.001;
Fig. 1A). A 50 mg/kg dose of GYY4137 produced antihyperalgesic ef-
fect from 1 to 2 h; RTL of 5.2 ± 0.3 s (n = 8) compared to 3.4 ± 0.3 s
(n = 6) for paclitaxel + vehicle-treated mice (p < 0.05; Fig. 1A) at 1 h,
and peaked and ended at 2 h with RTL of 7.1 ± 0.1 s (n = 8) compared
to 3.6 ± 0.2 s (n = 6) for paclitaxel + vehicle-treated mice (p < 0.01;
Fig. 1). The highest tested GYY4137 dose (100 mg/kg) also significantly
increased the RTL compared to paclitaxel + vehicle-treated mice from
1 to 4 h (when the experiment was terminated): RTL 5.1 ± 0.2 s (n = 8)
vs. 3.4 ± 0.3 s for paclitaxel + vehicle-treated mice (n = 6) at 1 h
(p < 0.05; Fig. 1A), peaked at 3 h (RTL 7.2 ± 0.3 s, n = 8 vs. 3.7 ± 0.2,
n = 6, respectively; p < 0.01; Fig. 1), and remained significantly high
until 4 h (RTL 4.7 ± 0.2 s, n = 8 vs. 3.1 ± 0.3, n = 6, respectively;
p < 0.01; Fig. 1A). A significant difference was observed in the ther-
apeutic effects of gabapentin 10 mg/kg and GYY4137 100 mg/kg only
against paclitaxel-induced thermal hyperalgesia at 3 h (RTL 5.8 ± 0.4 s,
n = 8 for gabapentin 10 mg/kg vs. 7.2 ± 0.3, n = 6 for GYY4137 100
mg/kg; p < 0.05). The other difference was in the starting time of in-
duction of antihyperalgesic effect, as GYY4137 100 mg/kg induced its
antihyperalgesic action earlier (after 1 h) than gabapentin (after 1.5 h),
and the effect was sustained for a longer time (up to 4 h) for GYY4137
compared to 3 h only with gabapentin 10 mg/kg (Fig. 1A).
Enzymatic H S synthesis in tissue homogenates was measured by
2
zinc trapping spectrophotometric assay as described previously [14]
with slight modification. Supernatant derived from the tissue homo-
genates (430 μl) was incubated with 20 μl pyridoxal 5′-phosphate (PLP,
2
mM) and the substrate L-cysteine (20 μl, 10 mM) and sealed with a
2
double parafilm layer to avoid leakage of H S gas generated after in-
cubating the tube in a 37 °C water bath for 45 min. Baseline controls
that contained trichloroacetic acid (TCA; 10 % w/v, 250 μl) were pre-
2
pared in parallel to obtain the basal H S background level. At the end of
the incubation period, zinc acetate (1% w/v, 250 μl) was injected to
trap the H S followed by TCA (10 % w/v, 250 μl) to terminate the
reaction. Subsequently, N,N-dimethylp-phenylenediamine sulfate dye
NNDPD; 20 mM, 133 μl) in 7.2 M HCl was added, followed by the
addition of FeCl (30 mM,133 μl) in 1.2 M HCl. After centrifugation
14,000 rpm for 4 min at 4 °C), absorbance (670 nm) of the resulting
2
(
3
(
methylene blue in the supernatant was measured using a 96-well mi-
croplate reader (Tecan Systems Inc., CA, USA) and compared against a
standard curve of NaHS (concentrations ranging from 3.125–250 μM).
At least 3 biological samples were assayed in duplicate and results were
expressed as nmol H
homogenate was estimated using Bradford assay (Bio-Rad, CA, USA).
2
S formed per mg protein. Protein level in each
2
.10. Anti-proliferative activity of GYY4137 and paclitaxel against breast
cancer cell lines
Human breast carcinoma cell line MCF-7 (American Type Culture
4
Collection, VA, USA) were seeded in a 24 well plate (∼10 ), allowed to
attach overnight and treated after 24 and 72 h with vehicle (DMSO),
various concentrations of paclitaxel (1 nM to 100 μM), GYY4137 (1 nM
to 100 μM) or a combination of both drugs. Growth was assessed by
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay (Promega, Madison, USA) after 96 h of incubation. The con-
centration of either drug that gave half-maximal response (IC50) was
calculated using non-linear regression analysis. The data were fitted to
a dose-response-inhibition equation (log [inhibitor] vs. normalized re-
sponse curve).
2.11. Statistical analysis
The software GraphPad Prism version 6.00 (GraphPad Software
Inc., USA) was used for data and statistical analyses. Statistical analyses
were performed using unpaired Student’s t test, one-way analysis of
variance (ANOVA) followed by Bonferroni’s multiple comparison post-
tests, Kruskal-Wallis test followed by Dunn’s multiple comparison test
and two-way repeated ANOVA followed by Bonferroni’s multiple
comparison post-tests. For Fig. 1A, which has several time points and
groups, the experimental design was such that data were arranged as
follows: each row represented a different time point and each column
represented a treatment group, so matched values were stacked into a
subcolumn. Based on this a repeated measures two-way ANOVA was
performed. This was followed by a post analysis for correction of
multiple comparisons with Bonferroni’s test (CIs and significance were
The optimal time point (2 h) and dose (50 mg/kg) for GYY4137
analgesic activity were selected based on the results from the dose- and
time-response experiment depicted in Fig. 1. The administration of the
K
ATP channel antagonist glibenclamide (10 mg/kg) to mice with pa-
clitaxel-induced thermal hyperalgesia did not alter the reaction latency
to hot-plate test compared to vehicle-treated mice (p > 0.05; Fig. 1B).
However, treatment with glibenclamide significantly attenuated the
antihyperalgesic effect of GYY4137 in the mice, i.e. a 37 % reduction in
reaction latency from 8.1 ± 0.3 s for paclitaxel + GYY4137 to
5.2 ± 0.1 s for paclitaxel + GYY4137 + glibenclamide (p < 0.01;
Fig. 1B). This result reveals that the acute antihyperalgesic action of
3

B. Qabazard, et al.
Biomedicine & Pharmacotherapy 127 (2020) 110210
Fig. 1. Antihyperalgesic effects of GYY4137against established paclitaxel-induced thermal hyperalgesia in female BALB/c mice is blocked by an ATP sensitive
potassium channel (KATP channel) inhibitor. (A) Effect of treatment with GYY4137 and gabapentin on mice with paclitaxel-induced thermal hyperalgesia in a hot-
plate test. Reaction latencies (seconds) were recorded from baseline values (taken before and at day 7 after first administration of paclitaxel) and at different time
points following treatment with gabapentin (10 mg/kg), GYY4137 (25, 50 and 100 mg/kg), or their vehicles. Each bar represents the mean ± S.E.M (n = 6-8). *
p < 0.05, ** p < 0.01 compared to paclitaxel vehicle, # p < 0.05 compared to gabapentin at the same time point after treatment. (B) Effect of treatment with
GYY4137 and glibenclamide, a KATP channel inhibitor, on mice with paclitaxel-induced thermal hyperalgesia in a hot-plate test. Reaction latencies (seconds) were
recorded from baseline values (taken before and at day 7 after first administration of paclitaxel) and after 2 h following treatment with glibenclamide (10 mg/kg),
GYY4137 (50 mg/kg), combination of GYY4137 and glibenclamide, or their vehicles. Each bar represents the mean ± S.E.M. (n = 9). *** p < 0.001 compared to
drug vehicle, ### p < 0.001 compared to GYY4137-treated group.
GYY4137 is dependent, at least partly, on KATP channels.
prophylactic effect of GYY4137.
3
.3. Effect of treatment with paclitaxel or paclitaxel plus GYY4137 on H
2
S
3
.2. Coadministration of paclitaxel with GYY4137 prevents the
level in plasma and H S generated by enzymes in tissues
2
development of paclitaxel-induced thermal hyperalgesia, cold allodynia and
mechanical allodynia
2
Plasma H S levels were not different in paclitaxel-treated group
compared to control (untreated) animals, but were significantly in-
creased in mice co-treated with paclitaxel plus GYY4137compared to
control animals or paclitaxel-treated group (p < 0.05; Fig. 3A) on day 7
after first drug administration. H S generated by enzymes in the brain
2
was similar in all the three groups i.e. control, paclitaxel-treated and
paclitaxel plus GYY4137 co-treated groups (p > 0.05; Fig. 3B) on day 7
Mice treated with paclitaxel plus GYY4137 (50 mg/kg) had RTL
8.22 ± 0.5 s, n = 10) similar to the vehicle-only treated control ani-
(
mals without hyperalgesia (9.29 ± 0.2 s, n = 8; p > 0.05; Fig. 3),
which were significantly higher than those of the mice treated with
paclitaxel alone. As such, the RTL of the mice co-treated with paclitaxel
plus GYY4137 at 50 mg/kg for 5 consecutive days was significantly
higher on day 7 after the first drugs administration than those treated
with paclitaxel alone, i.e. with hyperalgesia (RTL 8.22 ± 0.5 vs.
.67 ± 0.2 s, respectively, n = 10; p < 0.0001; Fig. 2A). Similar results
in mice co-treated with paclitaxel and GYY4137 (50 mg/kg) were ob-
served following assessment of cold and mechanical allodynia (Fig. 2B
C). In the cold plate test, the RTL of paclitaxel + GYY4137-treated
animals was significantly higher compared to paclitaxel-treated animals
RTL 50.09 ± 3.9 vs 26.04 ± 2.0 s, n = 8, respectively; p < 0.001;
Fig. 2B) and was similar to the vehicle-only treated group (RTL
5.86 ± 1.5 s, n = 8; p > 0.05; Fig. 2B). In a separate set of experi-
ments, paclitaxel + GYY4137 treated animals showed significantly
higher withdrawal threshold to mechanical stimuli than paclitaxel-
treated mice (3.27 ± 0.07 g vs 1.82 ± 0.08 g, respectively; n = 8;
p < 0.001; Fig. 2C), but was similar to the vehicle-only treated group
2
after first drug administration. On the other hand, H S generated by
enzymes in the spinal cord and paw skin was significantly lower in
paclitaxel-treated mice than in untreated control mice (p < 0.05;
Fig. 3C-D), and co-treatment with paclitaxel plus GYY4137significantly
5
increased H
2
S generation compared to paclitaxel only-treated mice
(
p < 0.05; Fig. 3C-D).
&
(
3.4. GYY4137 enhances the anti-proliferative effects of paclitaxel
5
The exposure of cultured breast cancer MCF-7 cells to paclitaxel (1
nM to 100 μM) for 96 h caused concentration-dependent decrease in
tumor cell viability as assessed by MTT assay (p < 0.05; Fig. 4A). Pa-
clitaxel inhibited MCF-7 cell proliferation by 40 % at 10 nM con-
centration which was increased to 50 % at concentrations ranging from
50 nM to 10 μM. At higher concentrations (50–100 μM) paclitaxel al-
most completely inhibited cell proliferation. The IC50 of paclitaxel was
80 nM. GYY4137 did not inhibit cell proliferation at concentrations
ranging from 1 nM to 50 μM, but significantly inhibited (40 %) cell
(
3.7 ± 0.1 g, n = 8; p > 0.05; Fig. 2C). These data show that the
concomitant treatment of paclitaxel with GYY4137 markedly prevented
the development of paclitaxel-induced thermal hyperalgesia as well as
cold and mechanical allodynia, thus indicating
a preventive/
4

B. Qabazard, et al.
Biomedicine & Pharmacotherapy 127 (2020) 110210
Fig. 2. Co-administration of GYY4137 with paclitaxel prevents the development of paclitaxel-induced thermal hyperalgesia, cold allodynia and mechanical allodynia
in female BALB/c mice. Effect of co-administration of GYY4137 with paclitaxel on the development of (A) paclitaxel-induced thermal hyperalgesia in a hot-plate test,
(
(
B) paclitaxel-induced cold allodynia in a cold-plate test and (C) paclitaxel-induced mechanical allodynia measured by a dynamic plantar aesthesiometer. Paclitaxel
2 mg/kg) was administered for 5 days with vehicle or GYY4137 (50 mg/kg). Each bar represents the mean ± S.E.M (n = 8-12). *** p < 0.001 compared to vehicles
only group (without hyperalgesia/allodynia) at the same time point after treatment. ### p < 0.001 compared to mice treated with paclitaxel-induced hyperalgesia/
allodynia treated with vehicle.
proliferation at 100 μM concentration (p < 0.05; Fig. 4A). GYY4137
100 μM) produced an antitumor effect in MCF-7 cell line, in agreement
with previously published reports [18]. The IC50 of GYY4137 was 373
μM. The combination regimen of paclitaxel (50 nM) plus GYY4137 (100
μM) significantly inhibited cell proliferation compared with mono-
therapy (Fig. 4B). GYY4137 increased the paclitaxel-induced decrease
of cell viability of this breast carcinoma cell line (Fig. 4B). The com-
bination of GYY4137 (100 μM) plus paclitaxel (50 nM) decreased the
tumor cell proliferation significantly much more that each individual
drug (p < 0.05; Fig. 4B). Thus, the combination of paclitaxel plus
GYY4137 does not negatively interfere with the anti-proliferative ac-
tivity of paclitaxel in breast cancer cell line in vitro; rather, produces a
(
Fig. 3. Measurement of H
of paclitaxel. (A) Plasma H
compared to both control (* p < 0.05) and paclitaxel only group (# p < 0.05). (B) Brain H
cord and (D) paw skin H S enzymatic production in paclitaxel-treated mice is significantly lower than untreated controls (* p < 0.05), and GYY4137 co-treatment
significantly increased spinal cord H
2
S level in control (untreated), paclitaxel-treated and paclitaxel and GYY4137 (50 mg/kg) co-treated mice at day 7 after first administration
S levels were not different in paclitaxel group compared to control animals, but were significantly increased with GYY4137 co-treatment
S enzymatic generation is similar in all the groups (p > 0.05). (C) Spinal
2
2
2
2
S generation compared to paclitaxel only-treated mice (# p < 0.05). Bars represent mean ± S.E.M of 6-8 samples.
5

B. Qabazard, et al.
Biomedicine & Pharmacotherapy 127 (2020) 110210
Fig. 4. Effects of monotherapy or combination regimen with paclitaxel or GYY on MCF-7 cell proliferation. Cells were seeded in a 24 well plate, allowed to attach
overnight and treated after 24 and 72 h with vehicle (solid bars) or various concentrations of (A) paclitaxel and GYY4137or (C) their combination. Growth was
assessed by MTT assay after 96 h of incubation. Each point or bar represents the mean ± SEM of 3 determinations. * p < 0.05 denote significant difference from
vehicle-treated group (0 in figure A), # p < 0.05 denote significant difference from paclitaxel monotherapy.
positive additive anti-proliferative effect.
blocker glibenclamide. Co-administration of GYY4137 with paclitaxel
prevented the development of paclitaxel-induced thermal hyperalgesia,
cold allodynia and mechanical allodynia. Paclitaxel-treatment induced
4. Discussion
2
a decrease in H S generation by enzymes in the paw skin and spinal
cord, which was prevented by co-administration with GYY4137.
GYY4137 had some anti-proliferative effects against a breast cancer cell
line MCF-7 and enhanced the anti-proliferative effects of paclitaxel
against these cells.
The antihyperalgesic effect of GYY4137 in mice with established
paclitaxel-induced thermal hyperalgesia was comparable to that of
2
The findings of the present study suggest that the slow release H S
donor GYY4137 has therapeutic potential and preventive role of in
CINP caused by paclitaxel in a murine model. GYY4137 produced an
acute antihyperalgesic effect comparable to the anticonvulsant gaba-
pentin, which is commonly used to relieve neuropathic pain. The an-
tihyperalgesic effect of GYY4137 was antagonized by the KATP channel
6

B. Qabazard, et al.
Biomedicine & Pharmacotherapy 127 (2020) 110210
gabapentin, although the highest dose of GYY4137 used (100 mg/kg)
produced a higher effect at one-time point (3 h) and had an earlier
onset and longer duration of antihyperalgesic activity than gabapentin
from 1 to 4 h versus from 1.5 to 3 h, respectively. Gabapentin has been
reported to ameliorate several types of acute and chronic pain [22]
including diabetic neuropathic pain [23] and CINP [24–27]. The results
of the current study demonstrate that GYY4137 has antihyperalgesic
activities against paclitaxel-induced thermal hyperalgesia. GYY4137
properties of paclitaxel against the MCF-7 cell line. These results sug-
gest that GYY4137 in addition to alleviation and prevention of pacli-
taxel-induced painful neuropathy it also enhances paclitaxel’s anti-
proliferative properties against a cancer cell line, and therefore may be
useful for the prevention and management of paclitaxel-induced painful
neuropathy.
5. Conclusion
2
and another H S donor NaHS have also been shown to relieve cold
allodynia induced by another anticancer drug oxaliplatin in mice [13].
NaHS also relieved cold allodynia induced by paclitaxel in mice [13].
Hydrogen sulfide donors have been reported to alleviate other types of
neuropathic pain such as neuropathic pain in chronic constriction in-
jured rats [10]. The activity of NaHS against oxaliplatin-induced cold-
allodynia was inhibited by the Kv7 potassium channel blocker XE991
In conclusion, the results of the current study reveal for the first
time that the slow-release H S donor GYY4137 can both prevent the
2
development of paclitaxel-induced neuropathic pain (PINP) and alle-
viate established PINP, while enhancing paclitaxel’s anti-proliferative
properties against a cancer cell line. These findings support previous
reports demonstrating the ability of H
hyperalgesia associated with other types of neuropathic pain in animal
models [9–12]. Deficiency in the generation of the endogenous H
seems to play a role in the pathogenesis of PINP. Further studies are
necessary to investigate the changes in H S generating enzymes and
their possible contribution to PINP or CINP in general. GYY4137 and
possibly other slow-release H S donors warrant further research as
2
S to ameliorate allodynia and
[
13], demonstrating a role of the voltage-gated Kv7potassium channels
in the antiallodynic effect of H S donors. There are many types of po-
2
2
S
tassium channels that fall under four classes: calcium-activated po-
tassium channels, inwardly rectifying potassium channels, tandem pore
domain potassium channels and voltage-gated potassium channels. The
involvement of potassium channels activation in mediating analgesia
has been demonstrated for several drugs, such as opioids [27], some
nonsteroidal anti-inflammatory drugs (NSAIDs) [28], and the anti-
depressants amitriptyline and clomipramine [29]. Previous studies in
2
2
potential drugs for prevention or alleviation of PINP in order to im-
prove the outcomes and quality of life for patients receiving paclitaxel
as a chemotherapeutic drug.
inflammatory pain models showed that the H
NOSH-aspirin) was superior to aspirin in reducing inflammatory pain
30], possibly due to H S inhibiting the production of pronociceptive
2
S-releasing aspirin
(
[
Declaration of Competing Interest
2
cytokines such as IL-1β, and directly blocking certain hyperalgesic
mediators in a mechanism dependent on modulation of KATP channels
There is no conflict of interests, financial or any other, in this study.
[
30]. Various studies have shown that H
activation of KATP channel, an inwardly rectifying potassium channel
31]. In the current study, the antihyperalgesic activity of GYY4137
against paclitaxel-induced thermal hyperalgesia was blocked by the
ATP channel blocker glibenclamide, demonstrating a role of KATP
channel in the antiallodynic effect of the H S donor. A similar effect was
also reported in the gastrointestinal tract as H S was shown to exert
antinociceptive activity by activating KATP channels [32].
S levels and expression of the H S-generating enzymes have been
2
S also produces its effects via
Acknowledgment
[
This study was funded by Kuwait University, Research
Administration, Project No. PT04/16. We also would like to acknowl-
edge "General Facilities Science (GF-S), Faculty of Science Nos. GS01/
K
2
03 (GC MS DFS - Thermo and Bruker 600 MHz NMR) and GS02/10 (LC-
2
MS/MS - Waters QToF)".
H
2
2
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