ORIGINAL ARTICLE

Synergistic antinociception between ZC88, an N-type voltage-

dependent calcium channel blocker, and ibuprofen in mouse
models of visceral and somatic inflammatory pain
W.-Z. Zhou*, T.-Y. Zhao*, Z.-Y. Wang, G.-Y. Lu, S.-Z. Zhang, C. Zhang, N. Wu, J. Li
Beijing Key Laboratory of Neuropsychopharmacology, State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of
Pharmacology and Toxicology, China

Correspondence Abstract
Ning Wu
E-mail: wuning7671@vip.126.com Background: A combination of analgesic agents with different
and mechanisms can induce additive or synergistic analgesia. The N-type
Jin Li voltage-dependent calcium channel (N-VDCC) is a novel therapeutic
E-mail: jinli9802@vip.163.com target for pain control. In addition to providing effective pain relief
when used alone, N-VDCC blockers produce synergistic analgesia when
*The two authors made equal contributions
used in combination with opiates. However, the interaction between N-
to this work.
VDCC blockers and nonsteroidal anti-inflammatory drugs (NSAIDs)
Funding sources remains unclear.
This work was supported by National Methods: Using isobolographic analysis and composite additive curve
Science and Technology Major Project for analysis, the antinociceptive interaction between ZC88, a selective N-
‘Major New Drugs Innovation & Develop- VDCC blocker and ibuprofen, a classical NSAID, was investigated in two
ment’ (No. 2014ZX09507003-004).
mouse models of visceral and somatic inflammatory pain.
Results: In the acetic acid writhing test, both ZC88 (10.5–42 mg/kg,
Conflicts of interest
None declared. intraperitoneally) and ibuprofen (50–200 mg/kg, orally) produced dose-
dependent antinociception, with ED50 values of 27.2 and 100.5 mg/kg,
respectively. ZC88 in combination with ibuprofen (ZC88 + ibuprofen)
Accepted for publication also induced significant antinociception, and isobolographic analysis
28 June 2018 revealed a synergistic interaction at 50% effect level. The experimental
ED50 (ED50 mix) of this combination (34.5 mg/kg) was significantly
doi:10.1002/ejp.1281
lower than the theoretical ED50 (ED50 add; 63.8 mg/kg). Additionally,
composite additive curve analysis displayed synergistic interaction at
other effect levels. In the formalin test, ZC88 or ibuprofen alone
significantly reduced late-phase rather than early-phase pain, with ED50
values of 31.3 and 123.9 mg/kg, respectively. Similarly, both
isobolographic analysis and composite additive curve analysis revealed
synergistic antinociception of ZC88 + ibuprofen (40.6 mg/kg of ED50 mix
vs. 77.6 mg/kg of ED50 add).
Conclusion: ZC88 in combination with ibuprofen produces synergistic
antinociception in mouse models of somatic and visceral inflammatory
pain.
Significance: Because ZC88 + ibuprofen achieves the same
antinociceptive effect at lower doses, the use of this combination could
result in fewer dose-related untoward effects. The potentiation of ZC88
on ibuprofen-induced antinociception indicates that N-VDCC blocker
has potential benefit to treat severe inflammatory pain.

46 Eur J Pain 23 (2019) 46--56 © 2018 European Pain Federation - EFICâ

W.-Z. Zhou et al.
1. Introduction
Pain complaints are the most prevalent symptoms,
which usually require analgesics for relief. Opiates
and nonsteroidal anti-inflammatory drugs (NSAIDs)
are commonly used for management of acute and
chronic pain. However, prolonged use of opiates is
limited by development of tolerance and depen-
dence, and use of NSAIDs is often accompanied by
gastrointestinal untoward effects. Therefore, new
analgesics or analgesic combinations are required to
relieve pain effectively with fewer untoward effects.
One promising target for the development of novel
analgesics is the N-type voltage-dependent calcium
channel (N-VDCC). N-VDCCs are almost exclusively
expressed in neuronal tissue and localized primarily
in presynaptic terminals to trigger neurotransmitter
release (Westenbroek et al., 1992; Gohil et al., 1994;
Wheeler et al., 1994). The input of peripheral nox-
ious stimuli activates the N-VDCCs in the nocicep-
tive Ad and C nerve fibre terminals located in the
superficial layers of the spinal cord dorsal horn,
which is the primary centre for transduction and
integration of pain signalling. Activation of these N-
VDCCs triggers the releases of glutamate, calcitonin
gene-related peptide (CGRP) and substance P, ulti-
mately inducing the sensation of pain (Bourinet
et al., 2014; Zamponi et al., 2015). Consequently,
blocking N-VDCCs results in the reduction of pain-
related neurotransmission, and thus produces anal-
gesia (Atanassoff et al., 2000; Smith et al., 2002).
Furthermore, N-VDCC knockout mice exhibit
reduced pain hypersensitivity in models of inflam-
matory and neuropathic pain (Hatakeyama et al.,
2001; Kim et al., 2001; Saegusa et al., 2001). There-
fore, N-VDCC is considered as a novel therapeutic
target for pain control.
Ziconotide, a 25-amino acid peptide, is an N-VDCC
blocker that has been approved for the treatment of
refractory severe pain such as cancer pain and
chronic neuropathic pain (Staats et al., 2004; Sch-
midtko et al., 2010; Pope and Deer, 2013). However,
intrathecal delivery and narrow therapeutic window
limit clinical use of ziconotide. Several newly devel-
oped small molecule blockers of N-VDCCs have
demonstrated antinociception in animal models
(Yamamoto and Takahara, 2009; Lee, 2014). In addi-
tion to providing pain relief when used alone, these
N-VDCC blockers, including peptide neurotoxins and
small-molecule compounds, produce synergistic
antinociception when used in combination with
morphine (Meng et al., 2008; Kolosov et al., 2011).
However, the interaction between N-VDCC blockers
© 2018 European Pain Federation - EFICâ
Synergistic antinociception of ZC88 and ibuprofen
and NSAIDs, another class of first-line drugs for pain
control, is not well characterized.
ZC88 (patent CN1884262B), a novel small-mole-
cule N-VDCC blocker, was designed and synthesized
by our group. Our previous study showed that ZC88
inhibited N-VDCC currents with an IC50 of
0.45 lmol/L, whereas it failed to block L-, P/Q- and
R-type calcium channels currents at concentrations
of up to 100 lmol/L (Zhang et al., 2015). ZC88 not
only produced antinociceptive activity when given
alone in thermal-stimulated acute pain, inflamma-
tory pain and peripheral neuropathic pain in rodent
models, but also potentiated morphine antinocicep-
tion in thermal-stimulated acute pain in mice (Meng
et al., 2008). However, the interaction between N-
VDCC blockers and NSAIDs has not been investi-
gated. Therefore, in this study, the antinociceptive
interaction between ZC88 and ibuprofen, a classical
NSAID, was determined in mouse models of acute
somatic and visceral inflammatory pain.
2. Materials and methods
2.1 Animals and drugs
Male Kunming (KM) mice, weighing 18–22 g, were
purchased from SPF (Beijing) Laboratory Animal
Technology Co., Ltd (Beijing, China). Animals were
housed under a 12-h light/dark cycle (lights on at
7:00 a.m. and lights off at 7:00 p.m.) at 22  1 °C
and 40–70% humidity, with food and water avail-
able ad libitum. All experiments were approved by
the Animal Care and Use Committee of Beijing Insti-
tute of Pharmacology and Toxicology, China, and
were carried out in compliance with the NIH Guide
for the Care and Use of Laboratory Animals. All the
studies were performed with double-blind designs.
ZC88 (synthesized by Beijing Institute of Pharma-
cology and Toxicology) and ibuprofen sodium salt
(Sigma-Aldrich, USA) were dissolved in normal sal-
ine. ZC88 and ibuprofen were administered via
intraperitoneal (ip) and oral (po) routes, respectively,
at a volume of 10 mL/kg.
2.2 Acetic acid writhing test in mice
This test was performed according to a previously
described protocol (Meng et al., 2008), with some
modifications. Mice were injected with 20 mL/kg of
0.6% acetic acid solution (ip). The number of wri-
thing during a 30-min period was counted, starting
5 min after the acetic acid injection. Antinociceptive
activity was expressed as a percentage of the
Eur J Pain 23 (2019) 46--56 47
Synergistic antinociception of ZC88 and ibuprofen
maximum possible effect (MPE%): MPE% = 100
(number of writhing for each mouse/average num-
ber of writhing in the control group) 9 100. ED50
values were calculated from the lg dose–response
curve by performing linear regression analysis.
In the test group, ZC88 (10.5–42 mg/kg, ip) or
ibuprofen (50–200 mg/kg, po) was administered 20–
30 min or 50–60 min before the acetic acid injection,
respectively; the mice in each control group received
normal saline (10 mL/kg, ip or po). To determine
the interaction between ZC88 and ibuprofen, we
administered different doses of ZC88 + ibuprofen
combined at a fixed-ratio of 1:1 (1/16, 1/8, 1/4, 1/2
and 3/4 of each ED50 dose). Ibuprofen (po) was
administered 30 min prior to ZC88 (ip) administra-
tion, and acetic acid was administered 20–30 min
after ZC88 administration.
2.3 Formalin test in mice
Before beginning of the test, each mouse was accli-
mated to the testing chamber for about 30 min indi-
vidually. Using a microsyringe, each mouse received
10 lL of a 5% formalin solution in normal saline
into the plantar surface of the right hindpaw (Saddi
and Abbott, 2000). Immediately after the formalin
injection, the mice were returned to the testing
chamber and pain behaviours were observed for
50 min. The duration of spontaneous licking or
flinching of the injected hindpaw was recorded dur-
ing the early phase I (0–5 min) and the later phase
II (10–50 min). The percentage of antinociception in
either phase I or phase II was calculated as follows:
Antinociception% = 100 (duration of hindpaw
licking or flinching for each mouse/average time of
hindpaw licking or flinching in the control
group) 9 100. The ED50 value was calculated as
mentioned above. In addition, formalin-induced paw
oedema was measured, which was calculated as fol-
lows: paw oedema (mm) = injected-paw thickness
1 h after formalin injection injected-paw thickness
before formalin injection (Shajib et al., 2008).
Drugs were administrated as described in the
acetic acid writhing test, except for ZC88 given alone
at dosages from 15 to 42 mg/kg.
2.4 Analysis of the interaction between ZC88
and ibuprofen
Using the method described by Tallarida (2000), we
performed isobolographic analysis to characterize the
interaction between ZC88 and ibuprofen in both the
acetic acid writhing test and the formalin test.
48 Eur J Pain 23 (2019) 46--56
W.-Z. Zhou et al.
Briefly, the experimental ED50 for the combination
of both drugs (ED50 mix) as well as the ED50 values
for ZC88 and ibuprofen alone were calculated from
their lg dose–response curves using linear regression,
respective, and then, the parallelism of the regres-
sion lines for ZC88 and ibuprofen alone was tested
using t-test. A theoretical ED50 (ED50 add) was calcu-
lated on the basis of the real ED50 values for each
drug under the assumption that only additive inter-
action occurs between the two drugs. The variance
of ED50 add (V(ED50 add)) was calculated as follows:
V(ED50 add) = f2 *V(ED50 ZC88) + (1 f)2 *V(ED50
Ibu), with f = 0.5. The significance of difference
between ED50 mix and ED50 add was analysed using t-
test. If the ED50 mix was significantly less than the
ED50 add, it was interpreted as a synergistic interac-
tion between these two drugs. If no significant dif-
ference was found between the ED50 mix and ED50
add, it was interpreted as an additive interaction.
An interaction index (c) was calculated using the
following formula: c = a/ED50 ZC88 + b/ED50 Ibu (Tal-
larida, 2002), in which a and b represent the respec-
tive doses of ZC88 and ibuprofen in the ED50 mix. If
c = 1, the interaction was considered additive, and if
c < 1, the interaction was considered synergistic.
Furthermore, to determine the interaction at other
effect levels, we used the composite additive curve
analysis (Tallarida, 2000), a more precise analysis
method. In brief, a composite additive regression line
was constructed by the additive set of points trans-
lated from the drug alone treatment. And the signifi-
cant difference between the composite additive line
and the experimental combination line was deter-
mined by F-distribution test.
2.5 Rotarod test in mice
The rotarod test was used to evaluate the effect of
the drugs on motor coordination or sedation (Stepa-
novic-Petrovic et al., 2008). One day before the test,
animals were trained on the rotarod at a constant
speed of 15 rpm three trials with 3 min/trial. Only
those mice that could stay on the rod for 180 s on
two consecutive trials were used in the following
test. On the testing day, the latency to fall was
recorded every 30 min after drug administration,
with 180 s as the cut-off latency. In single-drug
groups, mice were administered ZC88 (30 and
42 mg/kg, ip) or ibuprofen (100, 140 and 200 mg/kg,
po) and tested at 30, 60 and 90 min or 60, 90 and
120 min after drug administration, respectively. In
the ZC88 + ibuprofen combination groups (20.4 +
75.7 and 23.25 + 93 mg/kg), ibuprofen (po) was
© 2018 European Pain Federation - EFICâ
W.-Z. Zhou et al. Synergistic antinociception of ZC88 and ibuprofen

administered 30 prior to ZC88 (ip) administration.

The latency to fall was recorded for each mouse 30,
60 and 90 min after ZC88 administration.

2.6 Statistical analysis

The data are expressed as mean  standard error
of the mean (SEM) or as ED50 values with 95%
confidence intervals (CI). The data from the
antinociceptive activity and rotarod tests were anal-
ysed using one-way ANOVA followed by Bonfer-
roni’s test and two-way ANOVA with repeated
measurements followed by Bonferroni’s test,
respectively. The ED50 values (except for ED50 add)
were calculated from lg dose–response curves by
linear regression analysis. The parallelism of the lg
dose–response curves for ZC88 and ibuprofen
administered alone and the difference between
ED50 mix and ED50 add were analysed by t-test, as
described by Tallarida. Statistical significance was
defined as p < 0.05.

3. Results

3.1 Synergistic antinociception between ZC88

and ibuprofen in the acetic acid writhing test
in mice
Intraperitoneal injection of 0.6% acetic acid induced
visceral inflammatory pain in mice, as indicated by
the contraction of abdominal muscles accompanied
by extension of the forelimbs and elongation of the
body. Both ZC88 (10.5–42 mg/kg, ip) and ibuprofen
(50–200 mg/kg, po) significantly reduced the num-
ber of writhing in a dose-dependent manner (ZC88:
F(5,65) = 6.94, p < 0.001 by one-way ANOVA;
ibuprofen: F(5,66) = 12.15, p < 0.001 by one-way
ANOVA; Fig. 1A and B). According to the corre-
sponding lg dose–response curves (Fig. 2A), the ED50
values of ZC88 and ibuprofen were 27.2 and
100.5 mg/kg, respectively (Table 1). In addition, the
slopes of regression lines for ZC88 and ibuprofen
administered alone were 95.1  11.8 and
108.8  12.4, respectively, and statistical analysis
Figure 1 ZC88, ibuprofen and ZC88 + ibuprofen produced antinoci-
revealed the parallelism of their lg dose–response
ception in the acetic acid writhing test in mice. (A) ZC88 (intraperi-
curves (p > 0.05 by t-test). Therefore, normal type I
toneal), (B) ibuprofen (oral), (C) ZC88 + ibuprofen (intraperitoneal and
isobolographic analysis was performed in this oral). n = 11–12, mean  SEM. *p < 0.05, **p < 0.01 and
experiment. ***p < 0.001, one-way ANOVA followed by Bonferroni’s test.
ZC88 + ibuprofen also induced dose-dependent
antinociception in the acetic acid writhing test the lg dose–response curve was 34.5 mg/kg, which
(F(5,64) = 7.58, p < 0.001 by one-way ANOVA; was significantly lower than the theoretical ED50
Fig. 1C). Using the isobolographic analysis, the (ED50 add) value of 63.8 mg/kg (p < 0.05 by t-test;
experimental ED50 (ED50 mix) value calculated from Fig. 2B and Table 1). In addition, the interaction

© 2018 European Pain Federation - EFICâ Eur J Pain 23 (2019) 46--56 49

Synergistic antinociception of ZC88 and ibuprofen
Figure 2 Isobolographic analysis and composite additive analysis of
the antinociceptive interaction between ZC88 and ibuprofen in the
acetic acid writhing test in mice. (A) Linear regression of the lg dose–
response curves. n = 11–12, mean  SEM. (B) The isobologram for
ZC88 + ibuprofen. The ED50 values with 95% confidence intervals. ED50 mix,
experimental ED50 value; ED50 add, theoretical ED50 value; Ibu, ibupro-
fen. (C) Regression lines of the calculated composite additive points
and the experimental ZC88 + ibuprofen combination points.
index of ZC88 + ibuprofen was 0.54 (Table 1). These
results suggested the synergistic interaction between
ZC88 and ibuprofen at 50% effect level.
50 Eur J Pain 23 (2019) 46--56
W.-Z. Zhou et al.
Since the lg dose–response curve of ZC88 +
ibuprofen was significantly different from those of
drug alone, the composite additive curve analysis
was performed to further determine the interaction
at other effect levels. Fig. 2C showed the experimen-
tal ZC88 + ibuprofen combination line and the cal-
culated composite additive line. The F-distribution
test showed the significant difference between the
additive and the combination lines (F2,11 = 39.36,
which exceeded the tabular F value of 3.98 at 0.05
level), indicating the synergistic interaction between
ZC88 and ibuprofen.
3.2 Synergistic antinociception between ZC88
and ibuprofen in the formalin test in mice
Intraplantar injection of 5% formalin solution
induced a two-phase nociceptive response: the early
phase (Phase I) was induced within the first 5 min
and the late phase (Phase II) lasted from 10 to
50 min. In the early phase, neither ZC88 (15–
42 mg/kg, ip) nor ibuprofen (50–200 mg/kg, po) sig-
nificantly decreased the duration of hindpaw licking
and flinching (ZC88: F(4,55) = 1.657, p > 0.05 by
one-way ANOVA; ibuprofen: F(5,66) = 0.369,
p > 0.05 by one-way ANOVA; Fig. 3A and B). How-
ever, both ZC88 and ibuprofen dose dependently
reduced the formalin-induced nociceptive response
in the late phase (ZC88: F(4,55) = 2.98, p < 0.05;
ibuprofen: F(5,66) = 3.89, p < 0.01 by one-way
ANOVA; Fig. 3A and B), with ED50 values of 31.3
and 123.9 mg/kg, respectively (Fig. 4A; Table 2).
The slopes of the regression lines corresponding to
the lg dose–response curves for ZC88 and ibuprofen
administered alone were parallel (slope: 143.7 
12.1 vs. 93.5  17.3 of ZC88 vs. ibuprofen; p > 0.05
by t-test). In the late phase of the formalin test,
ZC88 + ibuprofen produced significant antinocicep-
tion (F(5,66) = 7.14, p < 0.001 by one-way ANOVA;
Fig. 3C), and the two compounds showed a synergis-
tic interaction at 50% effect level by the isobolo-
graphic analysis. The ED50 mix value of the
combination (32.8 mg/kg) was significantly lower
than the ED50 add value (73.3 mg/kg, p < 0.05 by t-
test; Fig. 4B; Table 2), and the interaction index
(0.52) was less than 1 (Table 2). Furthermore, the
composite additive curve analysis showed the syner-
gistic interaction at all effect levels (Fig. 4C;
F2,10 = 36.62, which exceeded the tabular F value of
4.10 at 0.05 level).
Additionally, ibuprofen (100, 140 and 200 mg/kg,
po) and ZC88 + ibuprofen (23.25 + 93 mg/kg,
ip + po) attenuated formalin-induced paw oedema,
© 2018 European Pain Federation - EFICâ
W.-Z. Zhou et al.
Table 1 Parameters of isobolographic analysis for ZC88 and ibuprofen combination in the acetic acid writhing test in mice.
Compound
ZC88
Ibuprofen
Compound combination Compound ratio
ZC88 + Ibuprofen 1:3.69
ED50 mix, experimental ED50 value; ED50 add, theoretical ED50 value; 95% CI, 95% confident interval.
whereas ZC88 (15–42 mg/kg, ip) had no effect on
the paw oedema (Fig. 3), indicating that ZC88 nei-
ther attenuated formalin-induced local oedema and
inflammatory nor enhanced ibuprofen’s action on
the local oedema and inflammatory.
3.3 Effects of ZC88 and ibuprofen in the
rotarod test in mice
In the rotarod test, ZC88 (30 and 42 mg/kg), ibupro-
fen (100, 140, and 200 mg/kg), and ZC88 + ibupro-
fen (20.4 + 75.7 and 23.25 + 93 mg/kg) did not
reduce the latency to fall (ZC88: Ftreatment (2,33) =
0.792, p > 0.05, Ftime (3,99) = 0.861, p > 0.05, Finteraction
(6143) = 1.069, p > 0.05; ibuprofen: Ftreatment (3,44) =
0.861, p > 0.05, Ftime (3,131) = 0.840, p > 0.05, Finteraction
(9,190) = 1.031, p > 0.05; ZC88 + ibuprofen: Ftreatment
(2,32) = 0.278, p > 0.05, Ftime (3,96) = 0.806, p > 0.05,
Finteraction (6,139) = 1.254, p > 0.05; by two-way
repeated-measures ANOVA; data not shown). This
suggested that the highest doses used in mice acetic
acid writhing test and formalin test could not cause
sedation or impair the motor coordination.
4. Discussion
A combination of two or more drugs with multiple
target mechanisms is commonly used to treat pain.
In the present study, we showed for the first time
that a combination of ZC88 and ibuprofen produced
synergistic antinociception in two mouse models of
acute visceral and somatic inflammatory pain: the
acetic acid writhing test and the formalin test.
In inflammatory pain, tissue damage or inflamma-
tion activates the peripheral nociceptive afferents
and the secondary sensory neurons in the dorsal
horn of the spinal cord (Millan, 1999). In the acetic
acid writhing test, the inflammation and injury
induced by acetic acid are associated not only with
local release of inflammatory factor prostaglandins
(PGs), particularly of the E series, resulting in the
activation of visceral peripheral nociceptive afferents,
© 2018 European Pain Federation - EFICâ
Synergistic antinociception of ZC88 and ibuprofen
ED50  SEM (95% CI)
27.2  1.7 mg/kg (22.4, 36.4)
100.5  5.6 mg/kg (83.0, 121.6)
ED50 mix  SEM (95% CI) ED50 add  SEM (95% CI) Interaction index
34.5  3.4 mg/kg (25.0, 49.3) 63.8  3.0 mg/kg (57.4, 71.0) 0.54
but also with release of excitatory glutamate, princi-
pally in the spinal cord (Giamberardino, 1999). Pre-
vious studies reported that N-VDCC activation
triggered the release of nociceptive neurotransmitters
and neurotransmodulators (e.g. glutamate, CGRP
and substance P) in the dorsal horn of the spinal
cord, and blocking N-VDCC resulted in the reduction
of pain-related neurotransmission (Atanassoff et al.,
2000; Smith et al., 2002; Bourinet et al., 2014; Zam-
poni et al., 2015). Additionally, NSAIDs, such as
ibuprofen, could inhibit peripheral cyclooxygenase 2
(COX-2) and reduce PGs synthesis to relieve inflam-
matory pain. Thus, ZC88 and ibuprofen reduced the
acetic acid-induced pain response likely via central
and peripheral mechanisms, respectively. In the for-
malin test, the two phases of the pain response are
considered to be associated, at least partially, with
distinct mechanisms (Hunskaar and Hole, 1987;
Haley and Dickenson, 2016). The early phase is due
to direct stimulation of the nociceptors of peripheral
C fibre nerve endings by formalin, resulting in the
high intensity of C fibres firing. In contrast, the late
phase is associated with formalin-induced release of
peripheral mediators such as PGs and bradykinin,
which causes the lower intensity of C fibre firing
accompanied by facilitation of dorsal horn neuronal
responses, reflecting the integration of peripheral
and central (spinal/supraspinal) signalling. Therefore,
by the blockade of PGs synthesis via inhibiting COX-
2, NSAIDs (e.g. ibuprofen), could relieve late-phase
rather than early-phase pain (Malmberg and Yaksh,
1995a; Tegeder et al., 2001), which is consistent
with the findings of the present study. As mentioned
above, since N-VDCC activation triggers the release
of nociceptive neurotransmitters and neurotrans-
modulators from the central terminals of primary
afferent neurons (Bourinet et al., 2014; Zamponi
et al., 2015), N-VDCC blockers may suppress both
early- and late-phase pain in the formalin test. In
the present study, however, systemic administration
of ZC88 significantly relieved formalin-induced
late-phase pain, with no significant reduction of the
Eur J Pain 23 (2019) 46--56 51
Synergistic antinociception of ZC88 and ibuprofen W.-Z. Zhou et al.

Figure 3 ZC88, ibuprofen and ZC88 + ibuprofen produced antinociception in the formalin test in mice. (A) ZC88 (intraperitoneal), (B) ibuprofen
(oral), (C) ZC88 + ibuprofen (intraperitoneal and oral). n = 11–12, mean  SEM. *p < 0.05, **p < 0.01 and ***p < 0.001, one-way ANOVA fol-
lowed by Bonferroni’s test.

nociceptive response in the early phase. Similarly,
intrathecal administration of ziconotide, a selective
N-VDCC peptide blocker, appeared to be more effec-
tive at suppressing the late phase rather than the
early phase of the behavioural response to formalin
(Malmberg and Yaksh, 1995b; Bowersox et al., 1996;
Wang et al., 2000). Additionally, similar to their
weaker effects during the early phase of the formalin
test, ziconotide and ZC88 were not very effective at
increasing the pain threshold in the models of more
intense acute pain, such as the hot plate and radiant
heat tests in rats and mice (Malmberg and Yaksh,
1995b; Scott et al., 2002; Meng et al., 2008). We
presumed that the weaker effect of N-VDCC blockers
on early-phase pain in the formalin test and ther-
mal-stimulated acute pain might be due to the par-
ticipation of other calcium channels subtypes in the
intense stimuli-triggered presynaptic calcium influx

52 Eur J Pain 23 (2019) 46--56 © 2018 European Pain Federation - EFICâ

W.-Z. Zhou et al.
Figure 4 Isobolographic analysis and composite additive analysis of the
antinociceptive interaction between ZC88 and ibuprofen in formalin test in
mice. (A) Linear regression of lg dose–response curves in the late phase
pain. n = 11–12, mean  SEM. (B) The isobologram for ZC88 + ibupro-
fen. The ED50 values with 95% confidence intervals. ED50 mix, experimental
ED50 value; ED50 add, theoretical ED50 value; Ibu, ibuprofen. (C) Regres-
sion lines of the calculated composite additive points and the experi-
mental ZC88 + ibuprofen combination points.
and neurotransmitter release in the dorsal horn of
the spinal cord. Indeed, both N-type and T-type cal-
cium channels are expressed in cell bodies of dorsal
© 2018 European Pain Federation - EFICâ
Synergistic antinociception of ZC88 and ibuprofen
root ganglion and afferent nerve terminals in the
spinal cord dorsal horn and play an important role
in pain signal processing, although N-type channels
contribute in a more exclusive and greater degree
(Choi et al., 2007; Zamponi et al., 2009, 2015;
Jacus et al., 2012; Garcia-Caballero et al., 2014).
When two compounds are administered at the
same time, synergistic, additive or antagonistic inter-
actions can occur. In preclinical studies, isobolo-
graphic analysis is considered the gold standard for
the evaluation of the specific characteristics of inter-
actions between two compounds at 50% effect level
(Berenbaum, 1989), and composite additive curve
analysis is a more precise analysis at all effect levels
(Tallarida, 2000). Upon performing these analyses,
the results revealed a synergistic interaction between
ZC88 and ibuprofen in both the acetic acid writhing
test and the formalin test. As mentioned above,
ZC88 and ibuprofen produced antinociceptive activ-
ity via different complementary pathways at central
and peripheral levels, respectively. Therefore, the
synergistic interaction between ZC88 and ibuprofen
could be explained by the involvement of different
targets and sites (spinal N-VDCC vs. peripheral COX-
2) in their antinociceptive effects against somatic and
visceral inflammatory pain in mice.
In addition to the pharmacodynamic interaction,
the pharmacokinetic interaction between two com-
pounds may also contribute to synergistic action. To
avoid the interference of gastrointestinal absorption
of the tested compounds, we administered ibuprofen
by the po route, and ZC88 by the ip route. However,
it is not known whether a metabolic interaction
exists between ZC88 and ibuprofen, or if ibuprofen
affects ZC88 transporting into the central nervous
system. Therefore, we could not rule out the contri-
bution of a pharmacokinetic interaction between
ZC88 and ibuprofen to their synergistic action. Nota-
bly, in the acetic acid and formalin tests, the slope of
the dose–response curve of ZC88 + ibuprofen was
lower than that of the individual compounds and
the interaction tended to an additive property at
higher effect level, indicating the existence of some
mechanisms that affected the antinociceptive action
at high dosages. The reason for these differences in
slopes needs to be investigated in future studies.
For drugs that display synergistic interaction,
lower doses of each drug can be used to achieve an
equal or better pharmacological action with fewer
untoward effects derived from individual compounds
(Tallarida, 2001). In particular, a combination of two
analgesics with different mechanisms of action might
be more beneficial because their individual
Eur J Pain 23 (2019) 46--56 53
Synergistic antinociception of ZC88 and ibuprofen
Table 2 Parameters of isobolographic analysis for ZC88 and ibuprofen combinations in the formalin test in mice.
Compound
ZC88
Ibuprofen
Compound combination Compound ratio
ZC88 + Ibuprofen 1:3.96
ED50 mix, experimental ED50 value; ED50 add, theoretical ED50 value; 95% CI, 95% confident interval.
untoward effects may be different. Ibuprofen caused
gastrointestinal untoward effects because it nonselec-
tively inhibited peripheral COX-1 activity (McGetti-
gan and Henry, 2000). In mice and rats, ZC88
produced central nervous system-related untoward
effects (our unpublished data) at over antinocicep-
tion doses, which were similar to those induced by
ziconotide. Thus, we presumed that ZC88 in combi-
nation with ibuprofen at high doses may cause gas-
trointestinal and central nervous system-related
untoward effects. Because of the synergistic antinoci-
ception, theoretically, the ZC88 and ibuprofen com-
bination is less likely to cause untoward effects
because the doses of the individual components are
lower than that used for monotherapy. In addition,
since ZC88 and ibuprofen have different untoward
effects, using them in combination is unlikely to
potentiate their individual side effects. In patients
with rheumatic arthritis, increasing doses of ibupro-
fen lead to increased incidence of gastrointestinal
untoward effects (McGettigan and Henry, 2000);
therefore, the maximal tolerated ibuprofen dose is
usually not enough to ameliorate the severe inflam-
matory pain sufficiently. The synergistic interaction
between ZC88 and ibuprofen indicated that this
combination may have potential benefit to treat sev-
ere inflammatory pain. In addition, we previously
reported that ZC88 showed no abuse potential in
mouse conditioned place preference model, and that,
when used in combination with morphine, it could
potentiate morphine-induced antinociception with
decreasing morphine tolerance and dependence
(Meng et al., 2008). Indeed, leconotide, a peptide
neurotoxin that blocking N-VDCC, also displayed the
synergistic antinociception with morphine in a rat
model of bone cancer pain (Kolosov et al., 2011). In
addition to the antinociceptive effect of ZC88
monotherapy on inflammatory and neuropathic pain
in rodent models, the potentiations of ZC88 on
antinociception produced by ibuprofen and
54 Eur J Pain 23 (2019) 46--56
W.-Z. Zhou et al.
ED50  SEM (95% CI)
31.3  1.1 mg/kg (26.3, 37.3)
123.9  8.3 mg/kg (82.4, 186.2)
ED50 mix  SEM (95% CI) ED50 add  SEM (95% CI) Interaction index
40.6  3.2 mg/kg (32.3, 51.1) 77.6  4.2 mg/kg (68.4, 88.1) 0.52
morphine, two first-line analgesic agents commonly
used in a clinical setting, support the utility of ZC88
as an effective analgesic candidate.
Since the acetic acid writhing test and the formalin
test in mice belongs to acute inflammatory pain
model, the present finding indicated that ZC88 in
combination with ibuprofen produced synergistic
interaction to acute inflammatory pain relief. Com-
plete Freund’s Adjuvant (CFA) is widely accepted to
induce chronic inflammation pain in rodents, and
we performed the CFA-induced chronic inflamma-
tory pain model in KM mice. Unfortunately, the KM
mice were too sensitive to CFA injection. Even 5-lL
injection of 1 mg/mL CFA produced severe mechani-
cal allodynia; and ZC88 (42 mg/kg, ip), ibuprofen
(200 mg/kg, po) and ZC88 + ibuprofen (21 + 100
mg/kg, ip+po) only showed mild antinociceptive
activity, when drugs either prophylactic or therapeutic
administration (Fig. S1 and Appendix S1). In fact,
our unpublished data showed that ZC88 and ibupro-
fen significantly produced antinociceptive activity in
CFA-induced chronic inflammatory pain in SD rats.
Thus, we suppose that the mouse strain of KM
mouse may be unsuitable to CFA-induced chronic
inflammatory pain model. The antinociceptive inter-
action between ZC88 and ibuprofen to chronic
inflammatory pain should be investigated in mice of
other strains or rats model. This is the limitation of
the present study.
In conclusion, we found that ZC88, a small-mole-
cule selective N-VDCC blocker, in combination with
ibuprofen, a classic NSAID, produced synergistic
antinociception in two mouse models of acute
somatic and visceral inflammatory pain. This finding
may expand our understanding of the properties of
N-VDCC blockers, which constitute a new class of
promising analgesic candidates. Additionally, our
findings suggest that N-VDCC blockers can be used
in combination with NSAIDs for effective pain man-
agement with fewer untoward effects because lower
© 2018 European Pain Federation - EFICâ
W.-Z. Zhou et al.
dosages of the individual drugs are required to
achieve the same analgesic effect.
Author contributions
N.W. and J.L. designed the research; W.Z.Z., T.Y.Z., Z.Y.W.
and G.Y.L. performed the research; C.Z. synthesized ZC88;
N.W. and S.Z.Z. analysed the data; N.W., G.Y.L. and J.L.
wrote the manuscript.
References
Atanassoff, P.G., Hartmannsgruber, M.W., Thrasher, J., Wermeling, D.,
Longton, W. et al. (2000). Ziconotide, a new N-type calcium channel
blocker, administered intrathecally for acute postoperative pain. Reg
Anesth Pain Med 25, 274–278.
Berenbaum, M.C. (1989). What is synergy? Pharmacol Rev 41, 93–141.
Bourinet, E., Altier, C., Hildebrand, M.E., Trang, T., Salter, M.W. et al.
(2014). Calcium-permeable ion channels in pain signaling. Physiol Rev
94, 81–140.
Bowersox, S.S., Gadbois, T., Singh, T., Pettus, M., Wang, Y.X. et al.
(1996). Selective N-type neuronal voltage-sensitive calcium channel
blocker, SNX-111, produces spinal antinociception in rat models of
acute, persistent and neuropathic pain. J Pharmacol Exp Ther 279,
1243–1249.
Choi, S., Na, H.S., Kim, J., Lee, J., Lee, S. et al. (2007). Attenuated
pain responses in mice lacking Ca(V)3.2 T-type channels. Genes Brain
Behav 6, 425–431.
Garcia-Caballero, A., Gadotti, V.M., Stemkowski, P., Weiss, N., Souza,
I.A. et al. (2014). The deubiquitinating enzyme USP5 modulates
neuropathic and inflammatory pain by enhancing Cav3.2 channel
activity. Neuron 83, 1144–1158.
Giamberardino, M.A. (1999). Recent and forgotten aspects of visceral
pain. Eur J Pain 3, 77–92.
Gohil, K., Bell, J.R., Ramachandran, J., Miljanich, G.P. (1994).
Neuroanatomical distribution of receptors for a novel voltage-
sensitive calcium-channel antagonist, SNX-230 (omega-conopeptide
MVIIC). Brain Res 653, 258–266.
Haley, J.E., Dickenson, A.H. (2016). Evidence for spinal N-methyl-d-
aspartate receptor involvement in prolonged chemical nociception in
the rat. Brain Res 1645, 58–60.
Hatakeyama, S., Wakamori, M., Ino, M., Miyamoto, N., Takahashi, E.
et al. (2001). Differential nociceptive responses in mice lacking the
alpha(1B) subunit of N-type Ca(2+) channels. NeuroReport 12, 2423–
2427.
Hunskaar, S., Hole, K. (1987). The formalin test in mice: Dissociation
between inflammatory and non-inflammatory pain. Pain 30, 103–
114.
Jacus, M.O., Uebele, V.N., Renger, J.J., Todorovic, S.M. (2012).
Presynaptic Cav3.2 channels regulate excitatory neurotransmission in
nociceptive dorsal horn neurons. J Neurosci 32, 9374–9382.
Kim, C., Jun, K., Lee, T., Kim, S.S., McEnery, M.W. et al. (2001).
Altered nociceptive response in mice deficient in the alpha(1B)
subunit of the voltage-dependent calcium channel. Mol Cell Neurosci
18, 235–245.
Kolosov, A., Aurini, L., Williams, E.D., Cooke, I., Goodchild, C.S.
(2011). Intravenous injection of leconotide, an omega conotoxin:
Synergistic antihyperalgesic effects with morphine in a rat model of
bone cancer pain. Pain Med 12, 923–941.
Lee, M.S. (2014). Recent progress in the discovery and development of
N-type calcium channel modulators for the treatment of pain. Prog
Med Chem 53, 147–186.
Malmberg, A.B., Yaksh, T.L. (1995a). Cyclooxygenase inhibition and
the spinal release of prostaglandin E2 and amino acids evoked by
paw formalin injection: A microdialysis study in unanesthetized rats.
J Neurosci 15, 2768–2776.
© 2018 European Pain Federation - EFICâ
Synergistic antinociception of ZC88 and ibuprofen
Malmberg, A.B., Yaksh, T.L. (1995b). Effect of continuous intrathecal
infusion of omega-conopeptides, N-type calcium-channel blockers, on
behavior and antinociception in the formalin and hot-plate tests in
rats. Pain 60, 83–90.
McGettigan, P., Henry, D. (2000). Current problems with non-specific
COX inhibitors. Curr Pharm Des 6, 1693–1724.
Meng, G., Wu, N., Zhang, C., Su, R.B., Lu, X.Q. et al. (2008). Analgesic
activity of ZC88, a novel N-type voltage-dependent calcium channel
blocker, and its modulation of morphine analgesia, tolerance and
dependence. Eur J Pharmacol 586, 130–138.
Millan, M.J. (1999). The induction of pain: An integrative review. Prog
Neurobiol 57, 1–164.
Pope, J.E., Deer, T.R. (2013). Ziconotide: A clinical update and
pharmacologic review. Expert Opin Pharmacother 14, 957–966.
Saddi, G., Abbott, F.V. (2000). The formalin test in the mouse: A
parametric analysis of scoring properties. Pain 89, 53–63.
Saegusa, H., Kurihara, T., Zong, S., Kazuno, A., Matsuda, Y. et al.
(2001). Suppression of inflammatory and neuropathic pain
symptoms in mice lacking the N-type Ca2+ channel. EMBO J 20,
2349–2356.
Schmidtko, A., Lotsch, J., Freynhagen, R., Geisslinger, G. (2010).
Ziconotide for treatment of severe chronic pain. Lancet 375, 1569–
1577.
Scott, D.A., Wright, C.E., Angus, J.A. (2002). Actions of intrathecal
omega-conotoxins CVID, GVIA, MVIIA, and morphine in acute and
neuropathic pain in the rat. Eur J Pharmacol 451, 279–286.
Shajib, M.S., Rashid, R.B., Ming, L.C., Islam, S., Sarker, M.M.R.
et al. (2008). Polymethoxyflavones from Nicotiana plumbaginifolia
(Solanaceae) exert antinociceptive and neuropharmacological effects
in mice. Front Pharmacol 9, https://doi.org/10.3389/fphar.2018.
00085.
Smith, M.T., Cabot, P.J., Ross, F.B., Robertson, A.D., Lewis, R.J.
(2002). The novel N-type calcium channel blocker, AM336,
produces potent dose-dependent antinociception after intrathecal
dosing in rats and inhibits substance P release in rat spinal cord
slices. Pain 96, 119–127.
Staats, P.S., Yearwood, T., Charapata, S.G., Presley, R.W., Wallace, M.S.
et al. (2004). Intrathecal ziconotide in the treatment of refractory
pain in patients with cancer or AIDS: A randomized controlled trial.
JAMA 291, 63–70.
Stepanovic-Petrovic, R.M., Tomic, M.A., Vuckovic, S.M., Paranos, S.,
Ugresic, N.D. et al. (2008). The antinociceptive effects of
anticonvulsants in a mouse visceral pain model. Anesth Analg 106,
1897–1903.
Tallarida, R.J. (2000). Drug synergism and dose-effect data analysis. (Boca
Raton: Chapman & Hall/CRC).
Tallarida, R.J. (2001). Drug synergism: Its detection and applications. J
Pharmacol Exp Ther 298, 865–872.
Tallarida, R.J. (2002). The interaction index: A measure of drug
synergism. Pain 98, 163–168.
Tegeder, I., Niederberger, E., Vetter, G., Brautigam, L., Geisslinger, G.
(2001). Effects of selective COX-1 and -2 inhibition on formalin-
evoked nociceptive behaviour and prostaglandin E(2) release in the
spinal cord. J Neurochem 79, 777–786.
Wang, Y.X., Gao, D., Pettus, M., Phillips, C., Bowersox, S.S. (2000).
Interactions of intrathecally administered ziconotide, a
selective blocker of neuronal N-type voltage-sensitive calcium
channels, with morphine on nociception in rats. Pain 84,
271–281.
Westenbroek, R.E., Hell, J.W., Warner, C., Dubel, S.J., Snutch, T.P.
et al. (1992). Biochemical properties and subcellular distribution
of an N-type calcium channel alpha 1 subunit. Neuron 9, 1099–
1115.
Wheeler, D.B., Randall, A., Tsien, R.W. (1994). Roles of N-type and Q-
type Ca2+ channels in supporting hippocampal synaptic transmission.
Science 264, 107–111.
Yamamoto, T., Takahara, A. (2009). Recent updates of N-type calcium
channel blockers with therapeutic potential for neuropathic pain and
stroke. Curr Top Med Chem 9, 377–395.
Eur J Pain 23 (2019) 46--56 55
Synergistic antinociception of ZC88 and ibuprofen W.-Z. Zhou et al.

Zamponi, G.W., Lewis, R.J., Todorovic, S.M., Arneric, S.P., Snutch, T.P. Supporting Information
(2009). Role of voltage-gated calcium channels in ascending pain
pathways. Brain Res Rev 60, 84–89. Additional supporting information may be found online in
Zamponi, G.W., Striessnig, J., Koschak, A., Dolphin, A.C. (2015). The
physiology, pathology, and pharmacology of voltage-gated calcium
the Supporting Information section at the end of the article.
channels and their future therapeutic potential. Pharmacol Rev 67,
821–870.
Figure S1. Antinociceptive activities of ZC88, ibuprofen
Zhang, S., Yang, L., Zhang, K., Liu, X., Dai, W. et al. (2015). ZC88, a and ZC88 in combination with ibuprofen in CFA-induced
novel N-type calcium channel blocker from 4-amino-piperidine chronic inflammatory pain in Kunming mice.
derivatives state-dependent inhibits Cav2.2 calcium channels. Brain Appendix S1. Materials and Methods.
Res 1605, 12–21.

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