Evaluation of the analgesic effects of ammoxetine, a novel potent serotonin and norepinephrine reuptake inhibitor
Acta Pharmacologica Sinica
Evaluation of the analgesic effects of ammoxetine, a novel potent serotonin and norepinephrine reuptake inhibitor
Ting-ting ZHANG 0 1
Rui XUE 0
Lei ZHU 1
Juan LI 1
Qiong-yin FAN 0
Bo-hua ZHONG 0
Yun-feng LI 0
Cai-ying YE 1
You-zhi ZHANG 0
0 State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology , Beijing 100850 , China
1 Department of Pharmacology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing 100005 , China
Aim: The selective serotonin (5-HT) and norepinephrine (NE) reuptake inhibitors (SNRIs) are commonly used for the treatment of neuropathic pain and fibromyalgia. Ammoxetine ((±)-3-(benzo[d] [1,3]dioxol-4-yloxy)-N-methyl-3-(thiophen-2-yl)propan-1-amine) has been identified as a novel potent SNRI. In this study, we evaluated the acute analgesic properties of ammoxetine in different animal models of pain, and examined the involvement of monoamines in its analgesic actions. Methods: The analgesic effects of ammoxetine were assayed using models of acetic acid- and formalin-induced pain in mice, neuropathic pain induced by sciatic nerve injury (SNI), chronic constriction injury (CCI) and reserpine-induced fibromyalgia pain in rats. The contents of 5-HT and NE in brain regions of fibromyalgia rats were measured using HPLC-ECD. In all the experiments, duloxetine was used as a positive control drug. Results: Oral administration of ammoxetine (0.625-10 mg/kg) or duloxetine (2.5-40 mg/kg) dose-dependently decreased the number of acetic acid-induced writhing and formalin-induced first phase and second phase paw licking time in mice. Oral administration of ammoxetine (2.5-10 mg/kg) or duloxetine (10 mg/kg) alleviated mechanical allodynia in SNI and CCI rats and thermal hyperalgesia in CCI rats. The antiallodynic effect of ammoxetine in CCI rats was abolished by pretreatment with para-chlorophenylalanine methyl ester hydrochloride (PCPA, a 5-HT synthesis inhibitor) or α-methyl-para-tyrosine methylester (AMPT, a catecholamine synthesis inhibitor). Oral administration of ammoxetine (30 mg/kg) or duloxetine (50 mg/kg) significantly attenuated tactile allodynia in rats with reserpine-induced fibromyalgia. In the fibromyalgia rats, administration of ammoxetine (10, 30 mg/kg) or duloxetine (30, 50 mg/kg) dose-dependently increased the levels of 5-HT and NE, and decreased the metabolite ratio of 5-HT (5-HIAA/5-HT) in the spinal cord, hypothalamus, thalamus and prefrontal cortex. Conclusion: Ammoxetine effectively alleviates inflammatory, continuous, neuropathic and fibromyalgia-related pain in animal models, which can be attributed to enhanced neurotransmission of 5-HT and NE in the descending inhibitory systems.
ammoxetine; duloxetine; SNRIs; analgesia; neuropathic pain; fibromyalgia; 5-HT; NE; descending inhibitory system
Tricyclic antidepressants (TCAs) have been used for years
for the treatment of neuropathic pain syndromes, including
diabetic neuropathy, postherpetic neuralgia and migraine
]. However, the multifarious side effects of
TCAs caused by targeting muscarinic, histaminergic and test and formalin test. Male Sprague-Dawley rats weighing
α-adrenergic receptors result in poor tolerability in chronic 180–200 g were used for sciatic nerve injury models, and rats
]. Selective serotonin reuptake inhibitors weighing 250–300 g were used for the fibromyalgia
experi(SSRIs) or norepinephrine reuptake inhibitors (NRIs) are less ments. The animals were group-housed under a 12-h light/
effective in relieving pain[
]. Selective serotonin and nor- dark cycle at room temperature (23±1 °C) with free access to
epinephrine reuptake inhibitors (SNRIs) have been the most food and water. All experiments were conducted according to
promising agents in modulating pain symptoms because of the National Research Council’s guidelines.
their balanced effects on 5-HT and NE and their better
The existing three SNRIs include venlafaxine, duloxetine
and milnacipran. Duloxetine is the most commonly used
SNRI that has been approved for the treatment of diabetic
neuropathic pain (DNP), fibromyalgia and chronic
]. Although acting on the same targets, these
drugs display some differences in their pharmacological
profiles, and these differences might be responsible for their
different clinical activities and limitations[
]. The development
of new SNRIs might provide the opportunity to obtain
compounds with improved clinical outcomes in patients suffering
from depression and pain.
Ammoxetine is the S-(–) isomer of 071031B ((±)-3-(benzo[d]
]dioxol-4-yloxy)-N-methyl-3-(thiophen-2-yl)propan1-amine) and was described as a novel 5-HT and NE reuptake
]. Our unpublished data proved that
ammoxetine is a potent and selective SNRI with weak affinity to the
DA (dopamine) transporter, monoamine oxidase, histamine,
cholinergic, and 16 other GPCR opioid receptors.
Ammoxetine could be detected in the plasma 5 min after being orally
administered and is quickly distributed to the brain.
Ammoxetine also displayed lower hepatotoxicity than duloxetine
(data not shown). In the present study, we investigated the
effects of ammoxetine on pain behavior induced by acetic acid
or formalin in mice. The analgesic effects of ammoxetine on
neuropathic pain and fibromyalgia were also assayed in sciatic
nerve injury (SNI)/chronic constriction injury (CCI) and
reserpine treated rats, respectively. Moreover, we examined the
involvement of monoamines in the mechanisms of the
analgesic effects of ammoxetine in CCI rats and reserpine-induced
Materials and methods
Animals were purchased from the Beijing Vital River
Laboratory Animal Technology Company (Beijing, China). Male KM
mice weighing 22–25 g were used for the acetic acid writhing
Duloxetine hydrochloride was purchased from Shanghai
Wandai Pharmaceutical Corporation, Ltd (Taizhou, China).
Ammoxetine was synthesized in our institute (Beijing Institute
of Pharmacology and Toxicology). Gabapentin,
para-chlorophenylalanine methyl ester hydrochloride (PCPA),
α-methylpara-tyrosine methyl ester (AMPT), reserpine hydrochloride,
5-HT, dopamine (DA), NE, 5-hydroxyindole-3-acetic acid
(5-HIAA), homovanillic acid (HVA) and
3,4-dihydroxyphenylacetic acid (DOPAC) were purchased from Sigma-Aldrich
(St Louis, MO, USA). Ammoxetine, duloxetine and
gabapentin were dissolved in distilled water. PCPA was suspended
in distilled water with 5% Tween-80. AMPT was dissolved
in saline. Reserpine was dissolved in glacial acetic acid and
diluted to a final concentration of 0.5% acetic acid with
distilled water. All drugs were administered subcutaneously (sc),
intraperitoneally (ip), or orally (po) by gavage at
concentrations of 1.0 mL/kg for rats and 10 mL/kg for mice. The doses
for each drug used in experiment are discussed below.
Acetic acid writhing test in mice
Separate groups of 12 mice were pretreated (po) with
duloxetine (2.5–40 mg/kg), ammoxetine (0.625–10 mg/kg) or the
vehicle and were injected (ip) 30 min later with 2% acetic
acid (solution in water) at a concentration of 10 mL/kg. Each
mouse was then housed in an individual clear plastic
observation chamber. The numbers of writhes were counted between
5 and 20 min after the acetic acid injection. A writhe was
defined as stretching of the hind limbs accompanied by a
contraction of the abdominal muscles[
Formalin test in mice
Separate groups of 10 mice weighing 22–25 g were
acclimatized to individual cubicles for at least 20 min prior to the
experiment. Mice were pretreated (po) with duloxetine (2.5–40
mg/kg), ammoxetine (1.25–20 mg/kg) or the vehicle and were
injected (sc) 30 min later with formalin (20 μL of a 5% solution
in saline) into the dorsal lateral surface of the right hind paw
using a 30-G needle. Observations started immediately after
the formalin injection, and nociception was quantified based
on the total paw licking time in seconds in the first phase (0–5
min) and the second phase (15–35 min)[
Spared nerve injury was induced in rats according to
Decosterd and Woolf [
]. Male Sprague-Dawley rats (180–200
g) were anesthetized with 7% chloral hydrate. After shaving
and cleaning the left hind leg, the sciatic nerve was exposed
rats 24 h and 1 h before ammoxetine or distilled water. The
mechanical allodynia thresholds in CCI rats were tested before
and after PCPA or AMPT administration. The acute
antinociceptive effects of ammoxetine were then determined.
at the level of trifurcation into the sural, tibial, and common
peroneal nerves. The branches of the tibial and common
peroneal nerves were tightly ligated and severed, keeping the sural
nerve intact. The overlying muscle and skin were closed with
silk thread. The same surgical operations were performed
in the sham group of animals, although the nerve was only
exposed and not cut. After recovery, the rats were housed
in groups of 3–5 individuals. Baseline mechanical allodynia
thresholds were determined prior to SNI surgery. Drug
evaluation was performed 10 d after SNI surgery, when mechanical
allodynia was near the maximum. The pre-drug withdrawal
thresholds were measured, and animals that displayed an
increased mechanical sensitivity of more than 40% of the base
values were allocated to groups equally to minimize the
differences in the average thresholds among the groups (n=10/
group). Rats received a single dose of ammoxetine (2.5, 5 and
10 mg/kg, po), duloxetine (10 mg/kg, po) or the vehicle. The
withdrawal thresholds were again determined at 30, 60, 120
and 180 min post drug administration.
Mechanical allodynia (von Frey hair test)
The cutaneous nociceptive threshold of the operated hind
paws was assessed according to a previous method reported
]. Briefly, rats were placed in test cages with a
metal-mesh floor and were allowed to habituate for at least
30 min. Von Frey filaments (Touch-Test® Sensory Evaluators,
NC12775-99, North Coast Medical Inc, San Jose, CA, USA) of
0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0 and 26.0 g were applied
to the plantar surface of the left hind paw. A positive or
negative response was defined as a paw withdrawal response from
the pressure of a filament or the lack of a response within
6 s, respectively. Initially, a 2.0-g force filament was used for
the nerve-injured rats, and a 6.0-g force filament was used
for the sham rats. If a positive response to a given filament
occurred, the next-smaller filament was then used. If a
negaCCI procedures tive response occurred, the next-larger filament was used. The
CCI surgery was performed according to the method of test continued until four responses were collected after the
Bennett and Xie[
]. Male Sprague-Dawley rats (180–200 g) first change in response. The tactile stimulus producing a 50%
were anesthetized with 7% chloral hydrate. After shaving likelihood of paw withdrawal threshold (PWT) was used to
and cleaning the left hind leg, the left common sciatic nerve calculate the cutaneous nociceptive threshold using an
adaptawas exposed at the level of the mid-thigh through the biceps tion of the Dixon up-down paradigm.
femoris. The nerve was separated from the adhering tissue
proximal to the sciatic trifurcation. Four chromic gut ligatures Thermal hyperalgesia (Plantar test)
(4/0) (JINHUAN, Shanghai, China) were tied loosely around Thermal hyperalgesia was assessed using the plantar test
the nerve, 1–2 mm apart. The same surgical operations were (Model 390 G; IITC Life Science Inc, Woodland Hills, CA,
performed in the sham group of animals except for the addi- USA) and a modified method of Hargreaves[
]. In brief, the
tion of the ligatures. The mechanical allodynia thresholds rats were habituated to an apparatus consisting of individual
were determined before and 14 d after the CCI surgery. Ani- Perspex boxes placed on top of a heated glass plate (30±1 °C).
mals that displayed an increased mechanical sensitivity of A mobile radiant heat source was positioned under the glass.
more than 40% of the base values were allocated to groups A focused beam of radiant light (active intensity of 30%) was
equally (n=10/group). Before drug administration, thermal used to heat the plantar surface of the hind paw. The
pawhyperalgesia was determined. The acute antinociceptive withdrawal latency was defined as the time taken by the rat to
effects of ammoxetine (2.5, 5 and 10 mg/kg, po), duloxetine (10 remove its hind paw from the heat source. The cut-off point
mg/kg, po) or gabapentin (60 mg/kg, po) were determined. was set at 20 s to prevent tissue damage. The injured hind
The mechanical allodynia thresholds were tested 30, 60, 120 paws were tested 3 times, and the average withdrawal latency
and 180 min after a single dose of drug administration, and was calculated and used for the analysis.
thermal hyperalgesia was tested 180 min post-drug
The effects of PCPA and AMPT on the antinociception of ammoxetine
The potential contribution of the serotonergic system to the
pharmacological effect of ammoxetine was investigated
using PCPA (an inhibitor of 5-HT synthesis), and the possible
involvement of the catecholaminergic system was examined
using AMPT (an inhibitor of catecholamine synthesis). PCPA
(150 mg/kg, ip) or 5% Tween-80 was administered to CCI
rats for three consecutive days before the administration of
ammoxetine (10 mg/kg, po) or distilled water. In a separate
group, AMPT (200 mg/kg, ip) or saline was injected into CCI
The potential effect of ammoxetine on motor performance
was examined in normal uninjured rats at doses of 2.5, 5 and
10 mg/kg and in reserpine-treated rats at 30 mg/kg using an
accelerating rotarod (TSE 337500, Germany) test. The rotarod
speed was increased from 4 to 40 r/min over a 300 s period,
with the maximum time spent on the rotarod set at 300 s. The
rats received two training trials on the first day (separated by
3–4 h) prior to drug testing for acclimatization purposes. On
the day of testing, a baseline response was obtained, and the
rats were grouped to minimize the differences among groups.
The rats received a single dose of duloxetine, ammoxetine or
vehicle and were tested 1 and 3 h later.
Induction of fibromyalgia by reserpine injection
Fibromyalgia was induced in rats following Nagakura’s
]. Generally, reserpine was subcutaneously injected
at a dose of 1 mg/kg once daily for three consecutive days. It
has been shown that reserpine treatment causes significant
tremors, hypokinesia and decreased body weight. Food
pellets were placed on the floor of the cage after reserpine
injection to allow for easier feeding. The effects of the drugs were
evaluated 5 d after the last injection of reserpine. The
predrug tactile response thresholds were measured, and rats with
a threshold of less than 5 g were selected and allocated equally
into 5 groups, each of which received ammoxetine (10 and 30
mg/kg, po), duloxetine (30 and 50 mg/kg, po) or the vehicle.
The response thresholds were again measured 0.5, 1, 2 and 4 h
after the administration of drugs or the vehicle.
Monoamine assays in brain regions in fibromyalgia rats by
The content of monoamines including 5-HT and its
metabolite 5-HIAA, NE, DA and its metabolites DOPAC and HVA
in tissues was analyzed using HPLC-ECD as previously
]. Fibromyalgia rats treated with ammoxetine and
duloxetine were euthanized immediately after the tactile test.
After the prompt removal of the brains and spinal cords, the
hypothalamus, thalamus and prefrontal cortex were dissected
out on an ice-cold dish. The samples were immediately
frozen and stored in liquid nitrogen until assayed. The samples
were weighed and homogenized in 0.4 mol/L perchloric acid
with 0.5 mmol/L Na2-EDTA and 0.01% L-cysteine (10 mL/mg
of tissue) and then centrifuged at 12 000 r/min for 30 min at
4 °C. The supernatant was filtered and injected into the HPLC
system, which consisted of a microbore reverse-phase column
(particle size 5 μm, 250 mm×4.6 mm), a Waters e2465 pump
(flow rate 1.0 mL/min) and an e2695 electrochemical detector
with a VT03 flow cell glassy carbon working electrode set at
700 mV (with respect to an Ag/AgCl reference electrode). All
HPLC components and software were purchased from Waters
Technologies (Shanghai, China). The mobile phase (pH 3.7)
consisted of 85 mmol/L citrate, 100 mmol/L sodium acetate,
0.9 mmol/L octyl-sodium sulfate, 0.2 mmol/L EDTA, and 15%
methanol. External standard curves were used for sample
quantitation based on the area under curve. The injection
volume was 50 μL.
The statistical analysis was performed using GraphPad Prism
(GraphPad Prism 5.0, version 2.0; GraphPad Software Inc, San
Diego, CA, USA). The data are expressed as the mean±SEM.
The data from the writhing and formalin tests and the
monoamine levels measured by HPLC were analyzed using
Student’s t-test or a one-way analysis of variance (ANOVA)
followed by Dunnett’s test. For data exhibiting unequal
variances, a Mann-Whitney U-test or a Kruskal-Wallis test
followed by Dunn's Multiple Comparison Test was applied. The
ED50 values were determined using a triple-parameter logistic
equation. The ED50 was defined as the dose that produced
50% of the maximum possible effect. For the data obtained
from the time-course measurement studies (SNI, CCI,
reserpine induced fibromyalgia), analyses were conducted using
two-way repeated-measures ANOVA followed by
Bonferroni’s post-hoc analysis. The data for the PWTs before and
after administration with PCPA or AMPT were compared by a
paired t-test. Probability values of less than 0.05 were
considered statistically significant.
Ammoxetine attenuated pain behavior induced by acetic acid or
In the acetic acid-induced writhing test, compared with
vehicle treatment, oral administration of ammoxetine (0.625–10
mg/kg) reduced the number of writhing events
(Kruskal-Wallis test, Kruskal-Wallis statistic=36.67, P<0.01, Dunn’s Multiple
Comparison Test, P<0.05 for 2.5 and 5 mg/kg, P<0.01 for 10
mg/kg). Duloxetine (2.5–40 mg/kg) reduced the number of
writhing events (Kruskal-Wallis test, Kruskal-Wallis
statistic=20.26, P=0.0011, Dunn's Multiple Comparison Test, P<0.05
for 20 mg/kg, P<0.01 for 40 mg/kg). The ED50 (±95% CL)
values of reversing the writhing behavior were 2.01 (0–7.06)
mg/kg for ammoxetine and 10.03 (6.94–13.10) mg/kg for
duloxetine (Table 1, Figure 2).
The analgesic effect of ammoxetine was further
evaluated by a formalin test. Ammoxetine (1.25–20 mg/kg) dose
dependently reduced the paw licking time in both the first and
the second phases (one-way ANOVA, first phase: F5, 54=5.40,
P=0.0004, Dunn’s Multiple Comparison Test, P<0.01 for 10 and
20 mg/kg; second phase: F5, 53=9.182, P<0.01, Dunn’s Multiple
Comparison Test, P<0.01 for 5, 10 and 20 mg/kg). Duloxetine
(2.5–40 mg/kg) also reduced both the first and the second
phases of the paw licking time (one-way ANOVA, first phase:
F5, 54=7.78, P<0.01, Dunn’s Multiple Comparison Test, P<0.01
for 20 and 40 mg/kg; second phase: F5, 53=14.67, P<0.0001,
Dunn’s Multiple Comparison Test, P<0.01 for 10, 20 and 40
mg/kg). The ED50 (±95% CL) values in reversing the licking
time for ammoxetine were 9.89 (6.55–13.23) mg/kg for the first
phase and 4.74 (2.84–6.64) mg/kg for the second phase. The
ED50 (±95% CL) values in reversing the licking time for
duloxetine were 19.65 (14.52–24.79) mg/kg for the first phase and
10.06 (9.13–11.00) mg/kg for the second phase (Table 1, Figure
Ammoxetine produced analgesia in pain induced by sciatic nerve
We investigated if ammoxetine exerts an analgesic effect on
neuropathic pain using models of SNI and CCI. The SNI rats
showed a significant decrease in the mechanical threshold
compared with the age-matched sham animals (treatment:
F5, 50=51.59, P<0.01; time: F4, 50=15.78, P<0.01; treatment×time:
F20, 50=3.74, P<0.01). Ammoxetine displayed a significant
painrelieving effect. Orally administered ammoxetine at a dose
of 5 mg/kg increased the PWT at the 30, 60, and 120 min time
points. Ammoxetine (10 mg/kg) significantly elevated the
PWT at 30, 60, 120 and 180 min post-administration.
Duloxetine (10 mg/kg) caused a significant reversal of allodynia at
60, 120 and 180 min post-administration (Figure 3A).
CCI induced tactile allodynia and thermal hyperalgesia in
rats; the mechanical threshold (treatment: F6, 63=23.25, P<0.01;
time: F4, 63=14.72, P<0.01; treatment×time:F24, 63=2.76, P<0.0001)
and thermal latency (treatment: F6, 58=10.91, P<0.01; time:
F1, 58=53.26, P<0.0001; treatment×time：F6, 58=4.16, P=0.0015)
were decreased in operated rats compared with the sham
group animals. Ammoxetine displayed a pain-relieving effect
on mechanical allodynia in CCI rats. Ammoxetine increased
the PWT at 30, 60, 120 and 180 min after administration at
doses of 5 and 10 mg/kg. A statistically significant increase
in the PWT was observed only at 120 min post-administration
of duloxetine (10 mg/kg). Considering the undefined
efficacy of duloxetine, gabapentin was used as the positive
control. Gabapentin (60 mg/kg) produced a significant reversal
of allodynia at 30, 60, 120 and 180 min post-administration
Ammoxetine (10 mg/kg) significantly increased the paw
withdrawal latency in the plantar test in CCI rats at 180 min
post-administration. Gabapentin (60 mg/kg) and duloxetine
(10 mg/kg) also produced significant reversals of thermal
hyperalgesia at 180 min post-administration (Figure 3C).
We tested the motor function using the accelerating rotarod
assay to eliminate the possibility that motor impairment
accounted for the analgesic effects of ammoxetine. The oral
administration of ammoxetine (2.5, 5 and 10 mg/kg) or
duloxetine (10 mg/kg) did not affect the latency in the rotarod
test in rats (treatment: F4, 35=0.06, P>0.05; time: F2, 105=11.01,
P<0.001; treatment×time: F8, 119=0.22, P>0.05) (Figure 3D).
Pretreatment with PCPA or AMPT abolished the pain-relieving
effect of ammoxetine
We evaluated the effect of pretreatment with PCPA (an
inhibitor of 5-HT synthesis) or AMPT (an inhibitor of
catecholamine synthesis) on the analgesia elicited by ammoxetine (10
mg/kg) in CCI rats. The administration of PCPA did not
modify the PWT (paired t-test, t=0.3363, P=0.74) in CCI rats
(Figure 4A1). Ammoxetine increased the PWT in CCI rats
pretreated with 5% Tween-80 (treatment: F1, 10=32.14, P<0.01;
time: F4, 50=3.31, P<0.05; treatment×time: F4, 59=6.09, P<0.01).
The effect of ammoxetine on the PWT in CCI rats pretreated
with PCPA was not significant (treatment: F1, 10=7.34, P>0.05;
time: F4, 50=0.67, P>0.05; treatment×time: F4, 59=0.65, P>0.05)
(Figure 4A2). The paired t-test showed that treatment with
AMPT had no significant effect on the PWT in CCI rats (paired
t-test, t=0.9778, P>0.05) (Figure 4B1). Ammoxetine increased
the PWT in CCI rats pretreated with saline (treatment:
F1, 10=16.96, P<0.01; time: F4, 50=3.86, P<0.01; treatment×time:
F4, 59=5.70, P<0.01). The effect of ammoxetine on the PWT in
CCI rats pretreated with AMPT was not significant (treatment:
F1, 10=0.06, P>0.05; time: F4, 10=0.69, P>0.05; treatment×time:
F1, 10=0.51, P>0.05) (Figure 4B2).
Analgesia effects of ammoxetine on fibromyalgia induced by
A previous study revealed that the withdrawal threshold
reached its lowest level at 4–7 d after the last injection of
reserpine (1 mg/kg once daily for three consecutive days)[
evaluated the pain relieving effects of the drugs five days after
the last injection of reserpine. Reserpine treatment
significantly decreased the PWT in rats compared with the healthy
controls (treatment: F5, 54=50.63, P<0.01; time: F4, 270=10.34,
P<0.001; treatment×time: F20, 299=1.51, P<0.01). Ammoxetine
at a dose of 30 mg/kg significantly increased the PWT at 60,
120 and 180 min post-administration. Duloxetine produced
a significant reversal of mechanical allodynia at a dose of 50
mg/kg at 60, 120 and 180 min post-administration (Figure 5A).
There was no difference in the motor performance between the
reserpine-treated rats and the healthy control rats (treatment:
F3, 20=0.23, P>0.05; time: F2, 60=1.28, P>0.05; treatment×time:
F6, 71=0.21, P>0.05). The administration of ammoxetine (30
mg/kg) or duloxetine (50 mg/kg) did not affect the motor ney test, U=0.000, P=0.0022; NE, Mann-Whitney test, U=0.000,
function in the reserpine-treated rats in the rotarod test (Figure P=0.0022; DA, Mann-Whitney test, U=0.000, P=0.0022). The
5B). 5-HIAA/5-HT and DOPAC/DA ratios in the reserpine-treated
rats were significantly increased relative to the control rats
Effects of ammoxetine on the levels of monoamines in the spinal in the spinal cord (5-HIAA/5-HT, Student’s t-test, t=8.883,
cord and brain in reserpine-treated rats P<0.0001), hypothalamus (5-HIAA/5-HT, Mann-Whitney test,
The levels of monoamine transmitters (5-HT, NE, DA) and U=0.000, P=0.0022; DOPAC/DA, Mann-Whitney test, U=0.000,
the metabolite ratios of 5-HT and DA (5-HIAA/5-HT and p=0.0022), thalamus (5-HIAA/5-HT, Mann-Whitney test,
DOPAC/DA) in the spinal cord and brain regions, including U=0.000, P=0.0022; DOPAC/DA, Mann-Whitney test, U=0.000,
the hypothalamus, thalamus and prefrontal cortex, in reser- P=0.0022) and prefrontal cortex (5-HIAA/5-HT, Student’s
pine-treated rats are summarized in Table 1. The measured t-test, t=12.55, P<0.0001; DOPAC/DA, Student’s t-test, t=3.072,
values of DA and DOPAC were lower than the minimum P=0.0118) (Table 2).
detection values in the spinal cord, and the chromatographic In the spinal cord, orally administered ammoxetine
peaks of HVA were not detectable in some samples in our increased the levels of NE (Kruskal-Wallis test, Kruskal-Wallis
detection system. Therefore, the DOPAC/DA values in the statistic=6.214, P=0.0205, Dunn’s Multiple Comparison Test,
spinal cord and levels of HVA were not determined. When P<0.05 for 30 mg/kg ammoxetine) and reversed the effects
compared with the healthy control rats, the levels of 5-HT, NE of reserpine on the 5-HIAA/5-HT ratio (one-way ANOVA,
and DA in reserpine-treated rats were significantly decreased F2, 15=32.76, P<0.001, Dunn’s Multiple Comparison Test, P<0.01
in the spinal cord (5-HT, Mann-Whitney test, U=2.000, for 10 mg/kg ammoxetine and P<0.01 for 30 mg/kg
ammoP<0.0317; NE, Mann-Whitney test, U=0.000, P=0.0022), hypo- xetine) in the reserpine-treated rats. Duloxetine at a dose of
thalamus (5-HT, Student’s t-test, t=11.42, P<0.01; NE, Student’s 50 mg/kg significantly increased the levels of 5-HT (one-way
t-test, t=7.163, P<0.01; DA, Student’s t-test, t=10.67, P<0.01), ANOVA, F2, 15=5.092, P=0.0205, Dunn's Multiple Comparison
thalamus (5-HT, Student’s t-test, t=17.67, P<0.01; NE, Mann- Test, P<0.05) and NE (one-way ANOVA, F2, 15=7.398, P=0.0058,
Whitney test, U=0.000, P=0.0043; DA, Mann-Whitney test, Dunn’s Multiple Comparison Test, P<0.01) and decreased
U=0.000, P=0.0022) and prefrontal cortex (5-HT, Mann-Whit- the 5-HIAA/5-HT ratio (Kruskal-Wallis test, Kruskal-Wallis
mg/kg ammoxetine). Ammoxetine significantly reversed
the effects of reserpine on the 5-HIAA/5-HT ratio (one-way
ANOVA, F2, 15=10.26, P=0.0016, Dunn’s Multiple Comparison
Test, P<0.01 for 10 mg/kg ammoxetine, P<0.01 for 30 mg/kg
ammoxetine). Duloxetine increased the levels of 5-HT
(oneway ANOVA, F2, 14=3.743, P=0.0499, Dunn’s Multiple
Comparison Test, P<0.05 for 30 mg/kg duloxetine) and NE (one-way
ANOVA, F2, 13=5.276, P=0.0210, Dunn’s Multiple Comparison
Test, P<0.05 for 50 mg/kg duloxetine) and decreased the
5-HIAA/5-HT ratio (Kruskal-Wallis test, Kruskal-Wallis
statistic=11.38, P=0.0034, Dunn’s Multiple Comparison Test, P<0.01
for 30 mg/kg duloxetine, P<0.05 for 50 mg/kg duloxetine)
In the prefrontal cortex, ammoxetine increased the levels
of 5-HT (one-way ANOVA, F2, 15=5.233, P=0.0189, Dunn’s
Multiple Comparison Test, P<0.05 for 10 mg/kg
ammoxetine, P<0.05 for 30 mg/kg ammoxetine) and NE (one-way
ANOVA, F2, 15=16.66, P=0.0002, Dunn’s Multiple
Comparison Test, P<0.01 for 10 mg/kg ammoxetine, P<0.001 for 30
mg/kg ammoxetine) and decreased the 5-HIAA/5-HT ratio
(one-way ANOVA, F2, 15=7.140, P=0.0066, Dunn’s Multiple
Comparison Test, P<0.05 for 10 mg/kg ammoxetine, P<0.01
for 30 mg/kg ammoxetine). Duloxetine increased the levels
of 5-HT (Kruskal-Wallis test, Kruskal-Wallis statistic=8.526,
P=0.0141, Dunn’s Multiple Comparison Test, P<0.05 for 30
mg/kg duloxetine) and NE (Kruskal-Wallis test,
KruskalWallis statistic=10.40, P=0.0055, Dunn’s Multiple Comparison
Test, P<0.01 for 50 mg/kg duloxetine) and decreased the
5-HIAA/5-HT ratio (one-way ANOVA, F2, 15=20.92, P<0.01,
Dunn’s Multiple Comparison Test, P<0.01 for 30 mg/kg
duloxetine, P<0.001 for 50 mg/kg duloxetine) (Table 2).
Neither ammoxetine nor duloxetine displayed any effect on
the levels of DA or on the DOPAC/DA ratio in the
hypothalamus, thalamus or prefrontal cortex in reserpine-treated rats.
statistic=10.71, P=0.0047, Dunn’s Multiple Comparison Test, In this study, ammoxetine displayed potent efficacy in
preP<0.01) (Table 2). venting acetic acid-induced visceral inflammatory pain and
In the hypothalamus, ammoxetine at a dose of 30 mg/kg formalin-induced continuous pain as demonstrated by lower
increased the levels of 5-HT (one-way ANOVA, F2, 15=3.915, ED50 values. Moreover, ammoxetine was efficacious in
relievP=0.0428, Dunn's Multiple Comparison Test, P<0.05) and ing neuropathic pain resulting from sciatic nerve injury (SNI
NE (one-way ANOVA, F2, 13=3.941, P=0.0459, Dunn's Mul- and CCI) and fibromyalgia induced by reserpine in rats.
Furtiple Comparison Test, P<0.05). Ammoxetine significantly thermore, the efficacy of ammoxetine in neuropathic pain
reversed the effects of reserpine on the 5-HIAA/5-HT ratio relief in the CCI and fibromyalgia models was dependent on
(one-way ANOVA, F2, 15=38.48, P<0.01, Dunn’s Multiple Com- 5-HT and NE.
parison Test, P<0.01 for 10 mg/kg ammoxetine, P<0.01 for 30 Acetic acid–induced writhing, which might be considered
mg/kg ammoxetine). Duloxetine increased the levels of 5-HT a model of visceral inflammatory pain, is a commonly used
(Kruskal-Wallis test, Kruskal-Wallis statistic=7.825, P=0.0200, pain test suitable for the evaluation of the action sites of
antiDunn’s Multiple Comparison Test, P<0.05 for 50 mg/kg nociceptive drugs at the supraspinal level. These results are in
duloxetine) and decreased the 5-HIAA/5-HT ratio (one-way accordance with previous studies that demonstrated that
antiANOVA, F2, 15=52.47, P<0.01, Dunn’s Multiple Comparison depressants, including fluoxetine, duloxetine and imipramine
Test, P<0.01 for 30 mg/kg duloxetine, P<0.01 for 50 mg/kg produced antinociceptive effects in the acetic acid-induced
duloxetine) (Table 2). writhing test in mice[
In the thalamus, ammoxetine significantly increased the lev- Previous studies identified the antinociceptive capacity of
els of NE (Kruskal-Wallis test, Kruskal-Wallis statistic=8.157, antidepressants, including the SNRIs milnacipran and
duloxP=0.0169, Dunn’s Multiple Comparison Test, P<0.05 for 30 etine, in relieving the pain induced by formalin[
]. The data
in the present study showed that ammoxetine and duloxetine
were efficacious in reducing paw licking time in the first and
second phases, with lower ED50 values in the second phase.
This finding is consistent with the data reported by Bardin
et al, which show that duloxetine and milnacipran reduced
formalin-induced nociceptive behavior in the two phases with
a more potent reduction in the second phase[
]. The second
phase of formalin-induced licking behavior is considered to
be pain associated with the function of spinal cord neurons,
which are under the regulation of the descending pain
]. Therefore, the analgesic effect of
ammoxetine and duloxetine might be due to the potentiation of 5-HT
and NE transmission in this model of pain. Lyengar et al
demonstrated that either the selective NRI thionisoxetine or the
SSRI paroxetine alone failed to reduce the late-phase licking
in the formalin pain model, even at a dose that significantly
elevated the neurotransmitters. However, lower doses of
paroxetine and thionisoxetine administered together resulted in
a statistically significant attenuation of the formalin-induced
]. These data suggest that the combined
increase of 5-HT and NE might be more beneficial in the
attenuation of persistent pain than an increase in either agent alone.
Ammoxetine significantly attenuated the neuropathic pain
30 min after administration in the SNI model, whereas the
effect of duloxetine (10 mg/kg) was detected at the time point
of 60 min. This finding indicated that ammoxetine had a
faster onset of analgesia than duloxetine in this neuropathic
pain model. In the CCI model of neuropathic pain, the acute
administration of ammoxetine significantly attenuated both
the mechanical allodynia and the thermal hyperalgesic
behavior. The potency of ammoxetine (10 mg/kg) was comparable
to that of gabapentin (60 mg/kg). Duloxetine at a dose of 10
mg/kg merely decreased mechanical allodynia in CCI rats at
120 min after administration. The results presented herein
indicate that ammoxetine is more efficacious in
suppressing neuropathic pain induced by CCI than duloxetine, as
evidenced by lower minimal effective doses. The analgesic
effects of SNRIs on neuropathic pain were estimated in
several animal models. However, discordant conclusions were
drawn from these studies. The study of Murai et al found that
duloxetine significantly ameliorated mechanical allodynia at a
dose of 30 mg/kg[
]. The study of Bomholt et al showed that
duloxetine (30 mg/kg) significantly attenuated thermal
hyperalgesia but failed to reduce mechanical allodynia in response
to von Frey hair stimuli in CCI rats[
]. The results from the
studies of Le Cudennec showed that duloxetine at a dose of
50 mg/kg significantly inhibited mechanical hyperalgesia and
heat hyperalgesia but not mechanical allodynia in CCI rats[
These discrepancies regarding the acute efficacy of duloxetine
on mechanical allodynia might be related to the experimental
protocols and the doses of the drug used in the various
We also evaluated the involvement of the serotonergic or
noradrenergic mechanisms underlying the antiallodynic effect
of ammoxetine using two monoamine-depleting agents in the
CCI model. PCPA, which inhibits the tryptophan hydroxylase
enzyme, has been reported to inhibit the synthesis of 5-HT and
deplete the endogenous stores of 5-HT in the central nervous
]. As shown in this study, the administration
of PCPA abolished the antiallodynic effect of ammoxetine.
This result indicates that 5-HT plays a critical role in the
pharmacological effect of ammoxetine. The results from Umut et
al demonstrated that PCPA administration did not reverse
the antiallodynic effects of mianserin, another SNRI agent, in
a diabetic neuropathy model of rats[
]. The difference in the
5-HT reuptake inhibition efficiency between ammoxetine and
mianserin might be correlated to these different results. It is
also possible that the mechanisms related to the
antinociceptive actions of antidepressants might differ between different
The relationship between NE and the effect of ammoxetine
was examined using AMPT, which selectively inhibits tyrosine
hydroxylase and decreases the synthesis of NE and DA. It has
previously been reported that the dual administration (with
a 23 h interval) of AMPT at 200 mg/kg induced a 50%–60%
reduction in the CNS NE level[
]. In the present study, we
found that AMPT treatment abolished the antiallodynic effects
of ammoxetine in CCI rats. In addition, our previous research
demonstrated that ammoxetine was a potent inhibitor of the
uptake of 5-HT and NE, whereas it had no effect on DA levels.
This finding suggests that the antiallodynic effect of
ammoxetine is at least partially related to a NE enhancement
mechanism. These studies indicate that a sufficient elevation of 5-HT
and NE might be required for effective pain relief by
antidepressants. The pronounced analgesic effect of ammoxetine on
neuropathic pain in rats might be attributed to its robust
inhibition of 5-HT and NE reuptake.
Fibromyalgia is a complex of syndromes and is difficult to
treat. Only three drugs, pregabalin, duloxetine and
milnacipran, have been approved for its treatment by the US FDA.
Duloxetine is recommended as the first choice for the
treatment of fibromyalgia[
]. However, there is limited evidence
for the efficacy of antidepressants in preclinical pain-models
of fibromyalgia. Although the exact molecular mechanisms
underlying the CNS sensitization and the painful symptoms of
fibromyalgia remain to be elucidated, evidence indicates that
the pathophysiology of fibromyalgia is related to the
dysfunction of biogenic amine-mediated pain control pathways in the
]. Nagakura et al developed a chronic-pain animal
model with a dysfunctional biogenic amine system induced by
reserpine that mimics fibromyalgia in humans. That group
reported that duloxetine at a dose of 30 mg/kg significantly
relieved the muscle pressure but not the tactile allodynia in
this model. In our present study, the acute administration of
ammoxetine (30 mg/kg) and duloxetine (50 mg/kg)
attenuated the tactile allodynia in the model established according
to the methods of Nagakura et al. Furthermore, we confirmed
that reserpine caused a significant decrease in biogenic amines
(DA, NE, and 5-HT) and increased the 5-HIAA/5-HT and
DOPAC/DA ratios in the spinal cord, hypothalamus,
thalamus and prefrontal cortex. These central nervous system
regions are deeply involved in descending inhibitory pain
signal processing. The administration of ammoxetine or
duloxetine increased the levels of 5-HT and NE, but not DA,
and decreased the 5-HIAA/5-HT ratio in the spinal cord and
brain regions. Under normal circumstances, SNRIs increase
the extra-cellular concentration of 5-HT and NE by blocking
the reuptake of these monoamines, although the total amount
of monoamines might not be changed significantly. The effect
of reserpine might make it clearer that SNRIs increase the
total content of 5-HT and NE by keeping these
neurotransmitters in the extra-cellular space and reducing their oxidation,
which could be supported by the decreased 5-HT metabolic
rate in the tissues. These results support the hypothesis that
the inhibition of 5-HT and NE reuptake might be responsible
for the antiallodynic effects of ammoxetine and duloxetine
in the fibromyalgia model. The data from the present study
and the study of Nagakura et al suggest that a high dose of
duloxetine is needed for significant antiallodynic effects in this
pain model. These results also suggest that strong inhibition
of 5-HT and NE reuptake is necessary for drug efficacy. This
study provides the evidence that SNRIs display antiallodynic
effects dependent on an increase in 5-HT and NE levels in the
descending inhibitory pathways in an animal model of
The present study suggests that ammoxetine produces more
potent analgesic effects than duloxetine in animal models of
inflammatory and continuous pain. Ammoxetine also had an
analgesic effect on neuropathic pain induced by SNI or CCI
and on pain associated with fibromyalgia induced by
reserpine. The pain-relieving mechanisms of ammoxetine could be
attributed to an increase in the transmission of 5-HT and NE
in the descending inhibitory pathways. Given the impressive
analgesic effects of ammoxetine, we consider the potential
effect and strategies directed to the treatment of neuropathic
pain and fibromyalgia.
5-HT, serotonin; NE, norepinephrine; SNRIs, selective
serotonin and norepinephrine reuptake inhibitors; 5-HIAA,
5-hydroxyindole-3-acetic acid; HVA, homovanillic acid;
DOPAC, 3,4-dihydroxyphenylacetic acid; SNI, sciatic nerve
injury; CCI, chronic constriction injury; TCAs, tricyclic
antidepressants; SSRIs, selective serotonin reuptake inhibitors; NRIs,
norepinephrine reuptake inhibitors.
This work was supported by the National Key New Drug
Creation Program (No 2012ZX09102101-004) and the National
Natural Science Foundation of China (No 81302761, 81274117,
Ting-ting ZHANG contributed to the research design,
performance of the experiments, data analysis and manuscript
writing. Rui XUE contributed to research design and
manuscript revision. Juan LI and Qiong-yin FAN participated in
the behavioral tests and manuscript revision. Bo-hua ZHONG
and Yun-feng LI contributed to the manuscript revision. Lei
ZHU helped in manuscript revision. You-zhi ZHANG and
Cai-ying YE contributed to the research design and
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