Analgesic Properties of Opioid/NK1 Multitarget Ligands with Distinct in Vitro Profiles in Naive and Chronic Constriction Injury Mice

Article abstract of DOI:10.1021/acschemneuro.7b00226

The lower efficacy of opioids in neuropathic pain may be due to the increased activity of pronociceptive systems such as substance P. We present evidence to support this hypothesis in this work from the spinal cord in a neuropathic pain model in mice. Biochemical analysis confirmed the elevated mRNA and protein level of pronociceptive substance P, the major endogenous ligand of the neurokinin-1 (NK1) receptor, in the lumbar spinal cord of chronic constriction injury (CCI)-mice. To improve opioid efficacy in neuropathic pain, novel compounds containing opioid agonist and neurokinin 1 (NK1) receptor antagonist pharmacophores were designed. Structure-activity studies were performed on opioid agonist/NK1 receptor antagonist hybrid peptides by modification of the C-terminal amide substituents. All compounds were evaluated for their affinity and in vitro activity at the mu opioid (MOP) and delta opioid (DOP) receptors, and for their affinity and antagonist activity at the NK1 receptor. On the basis of their in vitro profiles, the analgesic properties of two new bifunctional hybrids were evaluated in naive and CCI-mice, representing models for acute and neuropathic pain, respectively. The compounds were administered to the spinal cord by lumbar puncture. In naive mice, the single pharmacophore opioid parent compounds provided better analgesic results, as compared to the hybrids (max 70% MPE), raising the acute pain threshold close to 100% MPE. On the other hand, the opioid parents gave poor analgesic effects under neuropathic pain conditions, while the best hybrid delivered robust (close to 100% MPE) and long lasting alleviation of both tactile and thermal hypersensitivity. The results presented emphasize the potential of opioid/NK1 hybrids in view of analgesia under nerve injury conditions.

Full text of DOI:10.1021/acschemneuro.7b00226

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Article
Analgesic properties of opioid/NK1 multitarget ligands with distinct
in vitro profiles in naive and chronic constriction injury (CCI)-mice
Joanna Starnowska, Roberto Costante, Karel Guillemyn, Katarzyna Popiolek-Barczyk, Nga N. Chung,
Carole Lemieux, Attila Keresztes, Joost Van Duppen, Adriano Mollica, John M. Streicher, Jozef
Vanden Broeck, Peter W. Schiller, Dirk Tourwé, Joanna Mika, Steven Ballet, and Barbara Przewlocka
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00226 • Publication Date (Web): 12 Jul 2017
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Analgesic properties of opioid/NK1 multitarget ligands with distinct in vitro
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profiles in naive and chronic constriction injury (CCI)-mice
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Joanna Starnowska,† Roberto Costante,┴,$ Karel Guillemyn,┴ Katarzyna PopiolekꢀBarczyk, †
Nga N. Chung, ‡ Carole Lemieux, ‡ Attila Keresztes,§ Joost Van Duppen, ¥ Adriano Mollica, ◊
John Streicher,§ Jozef Vanden Broeck, ¥ Peter W. Schiller, ‡ Dirk Tourwé, ┴ Joanna Mika, †
Steven Ballet, ┴* Barbara Przewlocka†*
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†
Institute of Pharmacology, Department of Pain Pharmacology, Krakow, Poland
┴ Research Group of Organic Chemistry, Vrije Universiteit Brussel, Brussels, Belgium,
‡
Department of Chemical Biology and Peptide Research, Clinical Research Institute of
Montreal, Montreal, Canada,
§Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ,
U.S.A.
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¥
Animal Physiology and Neurobiology, Zoological Institute, KatholiekeUniversiteit Leuven,
Leuven, Belgium.
◊
Department of Pharmacy, “G. d’Annunzio” University, Chieti, Italy
$ Present address: IRBM Science Park s.p.a., Pomezia (RM), Italy
Corresponding authors
Prof. Barbara Przewlocka
Polish Academy of Sciences,
Institute of Pharmacology,
Department of Pain Pharmacology.
12 Smetna Street, 31ꢀ343 Krakow, Poland
Phone, 0048126623398; Eꢀmail: barbara.przewlocka@gmail.com
Prof. Steven Ballet
Research Group of Organic Chemistry,
Departments of Chemistry and Bioengineering Sciences,
Vrije Universiteit Brussel,
Pleinlaan 2, 1050 Brussel, Brussels, Belgium.
Phone: 0032ꢀ26293292; Eꢀmail: Steven.Ballet@vub.be
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ABSTRACT
The lower efficacy of opioids in neuropathic pain may be due to the increased activity of
pronociceptive systems such as substance P. We present evidence to support this hypothesis
in this work from the spinal cord in a neuropathic pain model in mice. Biochemical analysis
confirmed the elevated mRNA and protein level of pronociceptive substance P, the major
endogenous ligand of the neurokininꢀ1 (NK1) receptor, in the lumbar spinal cord of chronic
constriction injury (CCI)ꢀmice. To improve opioid efficacy in neuropathic pain, novel
compounds containing opioid agonist and neurokinin 1 (NK1) receptor antagonist
pharmacophores were designed. Structureꢀactivity studies were performed on opioid
agonist/NK1 receptor antagonist hybrid peptides by modification of the Cꢀterminal amide
substituents. All compounds were evaluated for their affinity and in vitro activity at the mu
opioid (MOP) and delta opioid (DOP) receptors, and for their affinity and antagonist activity
at the NK1 receptor. On the basis of their in vitro profiles, the analgesic properties of two new
bifunctional hybrids were evaluated in naïve and CCIꢀmice, representing models for acute and
neuropathic pain, respectively. The compounds were administered to the spinal cord by
lumbar puncture. In naïve mice the single pharmacophore opioid parent compounds provided
better analgesic results, as compared to the hybrids (max. 70% MPE), raising the acute pain
threshold close to 100% MPE. On the other hand, the opioid parents gave poor analgesic
effects under neuropathic pain conditions, while the best hybrid delivered robust (close to
100% MPE) and long lasting alleviation of both tactile and thermal hypersensitivity. The
results presented emphasize the potential of opioid/NK1 hybrids in view of analgesia under
nerve injury conditions.
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Keywords: Hybrid peptides; neuropathic pain; acute pain; NK1 antagonists; opioid agonism.
INTRODUCTION
Neuropathic pain is a chronic pain state which results from a lesion or pathology of the central
or peripheral nervous system; it affects up to 7ꢀ8% of the general population.3 Even though a
variety of agents are used to reduce pain in neuropathy, less than half of patients report
effective pain relief.4 However, one particular strategy, the design of drugs with biꢀ or even
multifunctional activity, offers a promising strategy to treat neuropathic pain. This strategy
addresses the complex nature of this pain type, which is associated with pathological
overactivation of endogenous pronociceptive systems as a maladaptive response to the
somatosensory system injury.5ꢀ6 Biꢀ or multifunctional agents are able to interact
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independently with two or more targets (such as different types of receptors) to achieve the
desired therapeutic effect.7ꢀ10 In particular, hybrid compounds designed as potential
therapeutic agents in neuropathic pain are able to activate the opioid system – to achieve an
antinociceptive effect – and hamper the activation of pronociceptive systems, to correct an
imbalance between antiꢀ and pronociception after repetitive opioid administration. In
consequence, such hybrids are expected to provide an efficacious analgesic effect with
lowered risk of adverse events.
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In previous studies on rats, we obtained promising results for hybrids acting as opioid
agonists and neurokininꢀ1 (NK1) receptor antagonists.2 In the present study we performed
biochemical analyses in mice under neuropathic pain conditions, which provide a basis for the
observations described above: the inefficacy of morphine to alleviate neuropathic pain is not
due to a reduced expression of the mu opioid peptide (MOP) receptor, but rather related to
counteraction due to an increased release of the pronociceptive substance P.11 This conclusion
motivates further studies to design new opioid agonist/NK1 receptor antagonists to treat
neuropathic pain, especially with regard to the influence of the relative potencies of the
included pharmacophores on in vivo efficacy. Therefore, in the current study, eight new
hybrids were prepared (compounds 6 – 13, RCCHM1-8, Table 1), and on the basis of their in
vitro pharmacological profile, the analgesic action of two of them in mice was compared to
that of a reference opioid 1 (AN81) and hybrid 3 (SBCHM1), both of which were tested
previously on rats.2 This comparison allowed a selection of the best hybrid structures for
testing of effective analgesia in mouse models of acute and neuropathic pain. Moreover, it
also provides insight into correlations between chemical structure and analgesic efficacy in
different behavioral tests.
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RESULTS AND DISCUSSION
Development of neuropathic pain and biochemical changes in the spinal cord, at a
selected time point after chronic constriction injury (CCI) to the sciatic nerve in mice.
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Development of neuropathic pain.
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A
B
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1
0
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Naive
CCI
***
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***
1
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7
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***
14
0
14
days after CCI
days after CCI
Figure 1. Development of hypersensitivity to tactile stimuli, as measured by the von Frey
test (panel A; cutꢀoff: 6 g) and thermal stimuli, as measured by the cold plate test (panel B;
cutꢀoff: 30 s) at different timeꢀpoints in naïve (black dotted line) and CCIꢀsubjected (red
line) mice
Twenty four hours after performing the chronic constriction injury (CCI), mice developed
hypersensitivity to tactile (von Frey test) and thermal (cold plate test) stimuli (Fig.1, panels A
and B, respectively). This was manifested as a significant decrease of the response threshold
in comparison with naive mice. Seven days after performing the nerve damage, CCIꢀmice
reacted to tactile stimuli (paw withdrawal) in the von Frey test at the mean force of 1.05 ± 0.3
g (cutꢀoff: 6 g). Tactile stimuli up to 6 g are neutral for healthy mice, and hence naive animals
do not react to it. Paw withdrawal of CCIꢀmice in the cold plate test was observed on average
after 5.6 ± 0.2 seconds (cutꢀoff: 30 s). Naive mice react after 26.9 ± 1.7 s on average. The
greatest manifestation of hypersensitivity symptoms was observed between the 7th and 14th
day after nerve injury (Fig. 1).
Changes in mRNA and protein levels of MOP, DOP and NK1 receptors, and in
substance P level in the lumbar spinal cord of mice at day 7 after CCI
To investigate biochemical changes that parallel behavioral hypersensitivity and clarify the
causes of the inefficacy of opioids to treat neuropathic pain, a quantification of the ꢀꢀopioid
(MOP), δꢀopioid (DOP) neurokinin 1 (NK1) receptors and substance P mRNA abundance and
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their protein level was performed using qRTꢀPCR and Western Blot methods in the lumbar
part of the spinal cord of mice, on the 7th day after CCI.
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mRNA
protein
1.3
MOP receptor
A
C
MOP receptor
B
D
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1.0
0.5
0.0
1.0
0.5
0.0
MOP receptor
DOP receptor
DOP receptor
2.0
1.5
1.0
0.5
0.0
*
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0.0
DOP receptor
NK1 receptor
NK1 receptor
E
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1.3
1.0
1.0
0.5
0.0
*
***
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0.5
0.0
NK1 receptor
2.0
substance P
substance P
1.5
1.0
0.5
0.0
*
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H
**
1.5
1.0
0.5
0.0
naive mice
contra
CCI mice
ipsi
Figure 2: The mRNA (left panel) and protein (right panel) level of MOP, DOP and NK1
receptors and substance P in lumbar spinal cord of CCIꢀexposed mice, on the 7th day after
surgical procedure. The data are presented as the mean ± SEM. Intergroup differences were
analyzed using ANOVA followed by Bonferroni’s multiple comparison tests. *p<0.05;
**p<0.01; ***p<0.001 indicate significant differences compared with naive mice.
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The qRTꢀPCR results on day 7 following CCI revealed no changes in MOP and DOP receptor
mRNA level in the spinal cord. Whereas the protein expression level was also not altered in
the case of the MOP receptor, DOP receptor protein (p < 0.05 vs control) levels increased
significantly on the injury site. A slight increase on the contralateral side did not reach
significance (Fig. 2 D). A decrease in the NK1 receptor mRNA level in the lumbar spinal
cord, on the ipsilateral (p < 0.001 vs control) and contralateral (p < 0.05 vs control) side was
observed. However, the protein level of NK1 receptor was significantly diminished only on
the ipsilateral side (p < 0.05 vs control) (Fig. 2 F). The level of substance P mRNA was
increased on the ipsilateral (p < 0.01 vs control), but not the contralateral side, which was
accompanied by a corresponding change in protein level (p < 0.05 vs control) (Fig. 2 G,H).
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The injuryꢀinduced development of neuropathic pain is clearly accompanied by changes in
mRNA and protein levels of both the opioid and neurokinin systems, as measured on day 7
after injury. It should be noted that there is also a report demonstrating an increase of SPꢀ
encoding gene TAC1 mRNA level in rats at day 3 after injury in the dorsal root ganglia
(DRG), without changes in the spinal cord.12 The changes demonstrated in our experiments on
mice support the pronociceptive activity of substance P. The increase in levels of both mRNA
and especially functionally active substance P on the injury side is balanced by a lower
expression of the NK1 receptor, seen as a lower level of the mRNA on both sides and of
protein on the lesion side. This indicates an increased activity of the pronociceptive system in
the development of injuryꢀinduced neuropathic pain. In contrast, there is a significant increase
of the DOP receptor protein on the ipsilateral side after damage. Moreover, neither mRNA
nor the protein of MOP receptor changes at the spinal cord level at day 7 after sciatic nerve
injury. Obara et al. did not observe any changes in mRNA of MOP in the spinal cord of CCIꢀ
rats at days 3 and 14 after injury.13 However, several lines of evidence have shown a
reduction of MOP receptor at the spinal cord level after nerve injury in rats and mice.14ꢀ15 The
observed discrepancies may result from the different methods of spinal cord dissection used to
collect lumbar part for biochemical analysis. In the mentioned work of Obara,13 no significant
changes in mRNA of DOP receptor (3 and 14 days after CCI) were observed, which is in line
with data presented here for day 7. Nonetheless, this report is the first to show that protein
levels of DOP receptor are elevated in the ipsilateral side of the spinal cord of CCIꢀmice 7
days after injury. In 2007 Kabli and Cahill observed an upꢀregulation of the DOP protein level
in DRG 14 days after sciatic nerve injury.16 It was suggested that this increase was correlated
with DOP trafficking.
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Our results support the hypothesis of altered NK1 system activation under neuropathic pain
conditions, which together with no changes in MOP receptor activity, increases the
importance of the pronociceptive system. Altogether, these findings provide the rationale for
the development of bifunctional opioid compounds containing an NK1 systemꢀtargeting
component as drug candidates for neuropathic pain treatment.
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Compound Design and Synthesis
As a starting point for the current study, bifunctional hybrids 3 (SBCHM1, HꢀDmtꢀ
DꢀArgꢀ
AbaꢀGlyꢀNMeꢀ3’,5’ꢀ(CF3)2ꢀBn) and 4 (KGCHM2, HꢀDmtꢀ ꢀArgꢀAbaꢀβꢀAlaꢀNMeBn),
D
which are a combination of an opioid agonist pharmacophore and NK1 receptor antagonist
pharmacophore to attenuate pronociceptive action, were selected.1ꢀ2 Both compounds have a
Nꢀmethylated amide group in the Cꢀterminal NK1 pharmacophore, which is essential for
affinity and antagonism at the NK1 receptor.1 Whereas secondary amide groups adopt
exclusively a trans conformation, tertiary amides exist as a mixture of cis and trans isomers
with a reduced activation energy for rotation.17 Since a large percentage of cis isomers (18ꢀ
22%) was found in an earlier study of NK1ꢀligands, this configuration might be important for
activity.18ꢀ19 Hence, the introduction of Nꢀalkyl groups larger than methyl may result in higher
populations of the cis amide bond. We previously showed that the introduction of an Nꢀ
isobutyl group resulted in a loss of affinity at the NK1 receptor.2 Therefore, the smaller
cyclopropyl group was evaluated in an attempt to avoid unfavorable steric interactions with
the NK1R (compounds 6 to 8, RCCHM1/2/8, Table 1). Additionally, a conformational
constraint was introduced by using the isoindoline amide (compounds 9 and 10;
RCCHM4/5). This leads to identical cis and trans isomers and reduces the number of
rotatable bonds, which could improve membrane permeability.20 Aside from the
bis(trifluoromethyl)benzyl substituent, the orthoꢀmethoxybenzyl group often occurs in small
molecule NK1 receptor antagonists,21 and hence this substituent was investigated as well.
Finally, because NK1 receptor antagonists have been shown to mostly possess two key
aromatic systems, which are in a stacked ‘L’ or ‘T’ configuration,21 we investigated whether a
change from the Cꢀterminal benzyl amide to naphtalenꢀ2ꢀylꢀmethyl amide could improve
stacking of the benzene ring within the aminoꢀbenzazepinone (Aba) scaffold with the larger
naphthalene ring (cf. compounds 12 and 13).
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The syntheses of the opioid tetrapeptide Dmtꢀ
DꢀArgꢀAbaꢀGlyꢀNH2 1 (AN81) and the
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reference hybrid 3 (SBCHM1) were reported previously.1 For the preparation of hybrids 6 –
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13, protected tetrapeptide analogs BocꢀDmtꢀDꢀArg(Pbf)ꢀAbaꢀGlyꢀOH and BocꢀDmtꢀDꢀ
8
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Arg(Pbf)ꢀAbaꢀβꢀAlaꢀOH were assembled as reported by a standard solid phase method.2
Next, the secondary amines were coupled to the two Bocꢀprotected tetrapeptide analogs using
DIC/HOBt. These secondary amines were prepared either by reductive amination of
cyclopropylamine with benzaldehyde, using sodium cyanoborohydride, or by alkylation of
cyclopropylamine with 2’,5’ꢀbis(trifluoromethyl)benzyl chloride, or of methylamine with 2ꢀ
(bromomethyl)naphtalene. The isoindoline amine was commercially available.
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Table1: Structure of the reference compounds 1 – 5 and of the new hybrids 6 – 13.
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Compound number (code)
n
R1
R2
1 (AN81)1
1
2
1
H
H
H
H
2 (KGOP1)2
3 (SBCHM1)1
4 (KGCHM2)2
2
5 (KGCHM5)2
6 (RCCHM1)
1
2
7 (RCCHM2)
8 (RCCHM8)
9 (RCCHM4)
10 (RCCHM5)
11 (RCCHM7)
2
1
2
1
1
12 (RCCHM3)
13 (RCCHM6)
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Binding and in vitro activity at the MOP, DOP and NK1 receptors
All compounds were evaluated for their affinity and in vitro activity at the MOP and DOP
receptors, and for their affinity and antagonist activity (except for the parent opioid 1and 2
compounds) at the NK1 receptor. The results are summarized in Table 2.
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Table 2: Binding and in vitro activity at the opioid and NK1 receptors. Results of the
compounds selected for in vivo testing are shown with a grey background.
NK1Ra NK1R GPIc MVDc MOPd DOPd
Compound
Structure
Ki
no.
IC50
(nM)
IC50
(nM)
Ki
Ki
b
pA2
(nM)
(nM) (nM)
1e
2f
HꢀDmtꢀDꢀArgꢀAbaꢀGlyꢀNH2
HꢀDmtꢀD ꢀArgꢀAbaꢀβꢀAlaꢀNH2
nd
nd
0.32
0.80
0.42
0.24
0.15
1.34
0.60
17.0
nd
nd
HꢀDmtꢀDꢀArgꢀAbaꢀGlyꢀNMeꢀ3’,5’ꢀ(CF3)2ꢀ
Bn
HꢀDmtꢀDꢀArgꢀAbaꢀβꢀAlaꢀNMeꢀBn
0.5
3e
7.80
6.44
nd
8.51
1.86
5.97
43.3
2.16
8.64
0.42
0.08
0.09
10.4
0.28
2.35
4f
5f
13.0
HꢀDmtꢀDꢀArgꢀAbaꢀGlyꢀNMeꢀBn
1530
HꢀDmtꢀ
(CF3)2ꢀBn
DꢀArgꢀAbaꢀβꢀAlaꢀNcPrꢀ3',5'ꢀ
735
5.36
18
16
47
190
6
HꢀDmtꢀ
HꢀDmtꢀ
HꢀDmtꢀ
HꢀDmtꢀ
D
ꢀArgꢀAbaꢀβꢀAlaꢀNcPrꢀBn
ꢀArgꢀAbaꢀGlyꢀNcPrꢀBn
520
95
5.20
5.50
0.25
3.16
4.84
19
10
5
6.6
11.2
2.1
2.7
66
68
16
81
26
7
8
D
0.30
0.20
0.06
0.90
D
ꢀArgꢀAbaꢀβꢀAlaꢀisoindoline
ꢀArgꢀAbaꢀGlyꢀisoindoline
>10.000
3495
501
9
D
7.3
5.0
8.0
10
11
HꢀDmtꢀ
HꢀDmtꢀ
D
ꢀArgꢀAbaꢀGlyꢀNMeꢀ2'ꢀOMeꢀBn
ꢀArgꢀAbaꢀβꢀAlaꢀNMeꢀ2'ꢀ
2.4
D
612
709
7.01
5.44
9.0
3.0
4.0
0.40
2.8
44
12
13
methylenenaphthyl
HꢀDmtꢀ ꢀArgꢀAbaꢀGlyꢀNMeꢀ2'ꢀ
methylenenaphthyl
D
10.0
228
a
Binding affinities of compounds for hNK1 receptors were determined by displacement of
[3H]substance P from binding sites in hNK1 receptors expressed in CHO cells. b The pA2 values were
c
calculated using Schild's equation.22 The GPI functional assay is representative of MOP receptor
d
activation, whereas the MVD is a DOP receptorꢀrepresentative assay.
Binding affinities of
compounds for MOP and DOP receptors were determined by displacement of [3H]DAMGO ([
Ala2,NMePhe4,Glyꢀol5]enkephalin) and [3H]DSLET ([ ꢀSer2,Leu5]enkephalinꢀThr6), respectively,
Dꢀ
D
from rat brain membrane binding sites.e,f Data from Ballet et al.1 and Guillemyn et al,2 respectively.
nd: not determined.
Our previous studies showed that when Gly4 (n = 1) in 3 was substituted by a βꢀalanine
residue (n = 2) and the bisꢀtrifluoromethyl substituents were removed to give 4, binding
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affinities to MOP and DOP receptors improved. Also, the activity in the GPI and MVD assays
(representative of MOP and DOP receptor agonism, respectively) improved in 4, but affinity
for the NK1 receptor and antagonism at the NK1 receptor was reduced (Ki shifted from 0.5
nM to 13nM and pA2 shifted from 7.80 to 6.44).2 Moreover, we had demonstrated that the
replacement of the Nꢀmethyl (R2) substituent by a bulky isobutyl substituent in either 3 or 4
resulted in a dramatic loss in NK1 receptor affinity.2 Here, we observe that replacement of the
Nꢀmethyl in the βꢀAlaꢀcontaining 4 by a Nꢀcyclopropyl group, which is smaller than isobutyl,
unfortunately also resulted in reduced binding and activity at the opioid receptors, especially
at DOP in 6 and 7. Concomitantly, binding affinity and antagonism at the NK1 receptor
substantially dropped. The same substitution in the Glyꢀcontaining analog 5,2 gave 8 with
somewhat reduced MOP receptor affinity (Ki= 0.3 versus 0.09 nM), but especially reduced
DOP receptor affinity (Ki= 68 versus 2.35 nM) and increased, but still rather low NK1 affinity
(Ki 95 versus 1530 nM) with a very modest pA2 value of 5.50. Additionally, the Nꢀmethylꢀ
benzyl substituent in 4 was replaced by an isoindoline group. This modification, leading to 9,
results in identical cis/trans isomers and limits the amount of rotatable bonds (nRot reduced
by two units),20 which could potentially improve binding and absorption. Surprisingly, it
resulted in a detrimental drop in NK1 antagonism (pA2: 6.44 to 0.25), while retaining good
activity and affinity at the opioid receptors. The glycine equivalent, 10, similarly has strongly
reduced NK1 receptor affinity and antagonism, but importantly it yielded a ligand with a
binding affinity for MOP receptor of 60 pM, while DOP receptor affinity was lower than that
of 9 (Ki of 81 vs 16 nM). Addition of a methoxyꢀgroup in orthoꢀposition of the Nꢀbenzyl
moiety in 11 did not result in good affinity and antagonism at the NK1 receptor, but again
good activity and affinity was observed at the opioid receptors. As mentioned above, the
insertion of larger aromatic groups might lead to increased aromatic stacking to provide the
relative ‘L’ꢀ or ‘T’ orientations of these groups, which was reported to be required for potent
antagonism at the NK1 receptor.21 The NK1 receptor antagonism of 12, containing a naphthyl
instead of a phenyl substituent, was in line with this hypothesis. Comparison of the Nꢀ
methylbenzyl 4 with the naphthyl derivative 12 indeed showed an increased pA2 (from 6.44 to
7.01), despite a lower NK1 receptor affinity, while retaining low nanomolar activity at the
opioid receptors. However, the Gly4 analogue 13 displayed a two orders of magnitude lower
NK1 receptor antagonism (pA2 of 7.01 vs. 5.44). It can be concluded that the nature of the Nꢀ
substituent in these hybrids is crucial for the NK1 receptor, whereas the opioid receptors are
much more tolerant to changes.
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Based on these results, the naphthyl substituted analogs 12 and 13 were selected for further in
vivo evaluation. Their properties were compared to the reference opioid parent 1 and hybrid 3.
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Behavioral study
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Effect of the selected hybrids and their parent compounds on motor performance after
i.t. administration in naïve mice
A dose of 1 µg/5µl per animal (i.e., ca. 1 nmol of hybrids 3, 12, 13, and 1.3 nmol for opioids 1
and 2) was chosen to assess its influence on motor performance of naïve mice in the RotaRod
test (Fig. 3). Vehicle, hybrids 3, 12, 13 and their reference opioid compounds 1 and 2 were
administered intrathecally; after 30 min, motor performance was assessed by the RotaRod
test. None of the hybrids influenced motor performance of naïve mice. In contrast, reference
opioid compounds 1 and 2 significantly hampered motor performance at the dose of ca. 1.3
nmol.
Motor performance - naive mice
30 min. after i.t. injection
300
200
***
100
***
0
Veh
1
3
2
12 13
Figure 3. The effect of intrathecal (i.t.) administration of ca. 1ꢀ1.3 nmol of reference opioid
compound 1 and its hybrid 3 and reference opioid compound 2 and its hybrids 12, 13 on
motor performance, as compared to Vehicle (Veh)ꢀtreated group, measured 30 min after
administration by the RotaRod test in naïve mice.
The antinociceptive effect of selected hybrids and their parent opioid compounds after
i.t. administration in naïve mice
The effect of hybrid 3 and its reference opioid 1, and hybrids 12 and 13, and their reference
opioid 2 on the pain threshold to a thermal stimulus was investigated in naive mice after
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administration to the spinal cord. Opioid agonist 1 was able to raise the latency of the
response to thermal stimuli (as measured by the tailꢀflick test) at a dose as low as 0.0013
nmol. At the highest dose tested (1.3 nmol), it provided a robust analgesic effect (close to
100% MPE) lasting up to 180 minutes after administration (Fig. 4A left panel). In contrast,
the effect of hybrid 3 proved to be weaker than its parent compound (Fig. 4B left panel).
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Opioid agonist 2 also gave longerꢀlasting analgesia at doses 0.1 – 1 nmol, though the effect
provided by the highest 1 nmol dose tested was no greater than 80% MPE (Fig. 4C left
panel). The two hybrids 12 and 13 gave longꢀlasting analgesic effects in acute pain, yet
weaker responses were noticed (60ꢀ70% MPE) in spite of raising the doses up to ca. 10 nmol
(Figure 4D,E left panel). This is in line with the weaker agonism at the opioid receptors of
the hybrids, compared to 1. Among all hybrids 13 was the one that provided the longest
lasting effect (p < 0.001 vs vehicle at 180 min after administration), even though the effect did
not rise above 50% MPE. (Fig. 4E left panel).
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Naive mice
CCI-exposed mice
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A
B
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1 (AN81)
1 (AN81)
***
**
100
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0.001
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1.3
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*
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0
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min
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min
min
G
3 (SBCHM1)
3 (SBCHM1)
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*
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***
0.9
9.5
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min
min
min
C
H
2 (KGOP1)
2 (KGOP1)
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min
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12 (RCCHM3)
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min
min
min
J
13 (RCCHM6)
13 (RCCHM6)
***
***
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***
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Veh
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0
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180
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180
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90
180
min
min
min
Figure 4. Effect of intrathecal (i.t.) administration of the reference opioid 1 and its hybrid 3
(A+F, B+G), and reference opioid 2 and its hybrids 12 and 13 (C+H, D+I, E+J) on pain
threshold as measured by the tailꢀflick test in naïve mice (left panel), and hypersensitivity as
measured by the von Frey and cold plate tests in CCIꢀexposed mice (right panel). The
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hypersensitivity tests were performed on mice (6 – 8 animals per group) between the 7thꢀ14th
day after chronic constriction injury (CCI). Interꢀgroup differences were analyzed by
ANOVA with Bonferroni’s Multiple Comparison PostꢀHoc Test. The results are presented as
percentages of the maximal possible effect (%MPE). The horizontal line in the figure
separates parents and hybrid compounds including Gly4 vs. βꢀAla4 residues. The doses are
presented in nmol/5µl/animal. *p < 0.05, **p < 0.01, ***p < 0.001 vs vehicleꢀtreated group
(Veh).
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The antinociceptive effect of selected hybrids and their parent compounds after i.t.
administration in CCI-subjected mice
All investigated compounds influenced the response latency in the neuropathic pain model.
As expected, the opioid structures 1 and 2 demonstrated a limited efficacy in both tests (Fig. 4
F,H right panel). Reference opioid 1 raised the response threshold in both tests, yet a weak
analgesic effect was observable ꢀ the response was not higher than 30% MPE in spite of
significant dose increments in the cold plate test, and not higher than 40% MPE in the von
Frey test (Fig. 4F right panel). In the case of compound 2, which is the reference opioid for
hybrids 12 and 13, no influence on the response threshold in the von Frey test was observed at
all. In addition, its analgesic action in the cold plate test was seriously limited, no more than
20 %MPE (Fig. 4H right panel).
Hybrid 3 was the weakest analgesic of the examined hybrids, but it was still able to raise the
response latency to ca. 60% MPE (Fig 4G right panel). Hybrid 13 proved to be the most
efficient analgesic in both tests. Application of the lowest tested dose (i.e. 0.1 nmol) raised the
response threshold to ca. 70% MPE in the von Frey test. This dose did not significantly
influence the response in the cold plate test, but raising the dose to 10 nmol resulted in a
robust analgesic effect (close to 100% MPE) for both tactile and thermal stimuli (Fig. 4J
right panel). The effect of hybrid 12 was weaker in both tests (Fig. 4I right panel).
Nonetheless, hybrid 3 was the only compound (p < 0.05) showing activity up to 180 min in
the von Frey test when administered at the moderate dose of approximately 1 nmol (Fig 4G
right panel). Hybrids 12 and 13, at the equivalent moderate dose, provided a shorter lasting
effect, not observable 180 min after administration (Fig. 4I,J right panel). Of note, 13 was
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the only compound that provided an effect at 180 min after administration in the cold plate
4
5
test (Fig. 4J, right panel), but only at the highest dose of 10 nmol.
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We then examined the effects of opioid parents and hybrid compounds in an acute pain model
in naïve mice. In contrast to the neuropathic pain results, the opioid parent structure 1 (AN81)
induced a superb analgesic effect in the acute pain model. It was efficacious at doses as low as
0.001 nmol/5µl/animal (p<0.05 vs vehicle) and it provided almost 100% MPE at the dose of 1
ꢀg. The effect of this dose was still very robust (>80% MPE) at 180 min after i.t.
administration. The opioid parent structure 2 demonstrated similar properties. However, both
of the reference opioids demonstrated significantly hampered action in the neuropathic pain
model: 1 exhibited a modest effect in the von Frey test (ca. 40% MPE at 30 min), where 2 was
not efficacious at all, and showed a poor effect in the cold plate test (<30%MPE). This effect
was no longer present 30 min after administration. This indicates that both 1 and 2 lose their
efficacy under neuropathic pain conditions, which stands in line with the results of previous
experiments on rats,2 as well as with the wellꢀdocumented phenomenon of limited analgesic
activity of opioids under neuropathic pain conditions.23ꢀ26 All hybrid compounds provided a
lowered analgesic effect (<70%MPE) in the tailꢀflick test up to 180 min after administration,
as compared to the parent opioids. Compound 3 was the strongest analgesic among all hybrids
at the highest dose 9.5 nmol/5µl/animal (p<0.001 at 30 min after administration). On the other
hand, hybrids 12 and 13 were able to provide an analgesic effect at the relatively low dose 0.1
nmol up to 180 min after administration. In contrast, 3 provided the weakest effect in the
neuropathic pain model, as compared to 12 and 13. At the highest dose 9.5nmol, hybrid 3 was
more potent in the cold plate test than in the von Frey test 30 min after administration. In the
von Frey test, 3 was the only compound that induced an analgesic effect at the dose 1µg (ca. 1
nmol) up to 180 min after administration.
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Summing up, the addition of the NK1 receptor antagonist pharmacophore to the opioid
sequence counteracted the loss of efficacy in a neuropathic pain model, as seen by the
differences in opioid parent structure activity in naive and neuropathic mice (Fig 4 A, C). It is
very interesting that hybrid 3 also loses its effectiveness, although its effect at a short time
after administration is close to that in naive mice. In contrast to hybrid 3, two hybrids of the
novel series (i.e. compounds 12 and 13) exhibit stronger effects in neuropathic pain than in
naive mice. Paradoxically, these two hybrids have a much weaker binding to the NK1
receptor compared to 3, with similar binding to the opioid receptors. It can be assumed that
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spinal NK1 receptors are less significant for that effect than receptors located in other
structures of the pain pathway, but this would require further investigation.
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Taking into consideration all behavioral results, 13 seems to exhibit the most beneficial
pharmacological profile as an analgesic drug: it provides a moderate (~60% MPE) effect in
attenuating acute pain, as measured in three time points after administration. In addition, its
analgesic effect in CCIꢀexposed mice is highly efficacious. It produced a robust response in
both the von Frey and cold plate tests, with the effect of the highest dose (10
nmol/5µl/animal) close to 100% MPE in both tests.
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As observed before, the best analgesic compounds have high MOP/DOP receptor agonist
potency and efficacy, but to be an effective analgesic for neuropathic pain it must be
combined with an NK1 receptor antagonist. In contrast to the MOP/DOP potency and
efficacy, NK1 potency and efficacy does not necessarily need to be very high.2, 27 The same
applies to motor performance, wherein the hybrids do not show impaired motor performance
as clearly manifested for the parent compounds. It seems that for both these opioid effects the
NK1 component is important, but not the strength of its (in vitro) antagonism. This is
important information when searching for new analgesics to be used for neuropathic pain
treatments.
CONCLUSIONS
In the present study we demonstrated the analgesic effect of bifunctional hybrids, both in
naïve mice and in a neuropathic pain mouse model. Our results support the concept of altered
NK1 system activation under neuropathic pain conditions which, in parallel with no changes
in MOP receptor mRNA level, increases the importance of the NK1 pronociceptive system.
These findings justify the strategy to use opioid compounds, containing an NK1 systemꢀ
targeting component.
We have studied the influence of the Nꢀterminal amide substituents in opioid agonist – NK1
receptor antagonist hybrids. Whereas these substituents do not dramatically influence the
affinity and agonist activity at the MOP and DOP receptors, their influence on NK1 receptor
affinity and antagonism is pronounced. The isoindolineꢀsubstituted analogs 9 and 10 are very
potent MOP/DOP receptor agonists, but their activity at the NK1 receptor is abolished. The
NMeꢀ2ꢀmethylenenaphthyl analogs 12 and 13 have the best balance of opioid agonism/NK1
receptor antagonism as determined in the in vitro study.
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As a consequence, we assessed the analgesic effects of three distinct opioid agonist – NK1
receptor antagonist hybrid compounds 12, 13 and 3, after intrathecal (i.t.) administration in
mice in an acute and neuropathic pain model. Each of the tested compounds demonstrates
unique analgesic properties, as determined in the different tests. Two hybrids of the novel
series (i.e. compounds 12 and 13) exhibit stronger effects in the neuropathic pain models, as
compared to an antinociceptive assessment in naive mice. Paradoxically, these two hybrids
have a much weaker NK1 receptor binding affinity as compared to the initial lead structure 3,
with similar binding affinities at the opioid receptors. It can be suggested that spinal NK1
receptors are less significant for that effect than receptors located in other structures of the
pain pathway, but this would require further investigation. As previously demonstrated, the
combination of a MOP/DOP agonist with a NK1 antagonist in a bifunctional compound can
delay or suppress analgesic tolerance,28ꢀ29 and transport an otherwise nonꢀpermeable NK1
antagonist into the brain.2 In addition, the present results demonstrate that weak NK1
antagonism is sufficient for potent analgesia in neuropathic pain models. This avoids the use
of high doses of a NK1 antagonist alone, which has been shown to induce neurotoxicity in
rats.27
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In brief, the addition of the NK1 receptor antagonist pharmacophore to the opioid sequence
counteracted the loss of efficacy in a neuropathic pain model, as seen in differences in opioid
parent structure activity in naive and neuropathic mice. Altogether, these findings justify a
continued search for dual opioid agonistsꢀNK1R antagonists, which in our hands emerged in
the discovery of compound 13, a compound which is efficacious in both acute and
neuropathic pain models in mice.
MATERIALS AND METHODS
General
Thinꢀlayer chromatography (TLC) was performed on glass plates precoated with silica gel
60F254 (Merck, Darmstadt, Germany) using the mentioned solvent systems. Purification of
organic molecules was done with flash chromatography (Davisil LC60A, 40ꢀ63 µm). Mass
Spectrometry (MS) was performed on a Micromass QꢀTof Micro spectrometer with
electrospray ionisation (ESI). Data collection and spectrum analysis was done with Masslynx
software. Analytical RPꢀHPLC was performed using a Waters 717plus autosampler, a Waters
1525 binary HPLC pump and a Waters 2487 dual absorbance wavelength detector (Milford,
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MA), on a Grace (Deerfield, IL) Vydac RP C18 column (25 cm x 4.6 mm x 5 ꢁm) using UV
detection at 215 nm. The mobile phase was a mixture of water and acetonitrile both
containing 0.1% TFA. The used gradient runs from 3 to 100% acetonitrile in 20 minutes at a
flow rate of 1 mL/min. Preparative RPꢀHPLC purification was done on a Gilson (Middleton,
WI) HPLC system with Gilson 322 pumps, controlled by the software package Unipoint, and
a reversed phase C18 column (Discovery®BIO SUPELCO Wide Pore C18 column, 25 cm x
2.21 cm, 5 ꢁm) using a gradient that increased by 1%/min of acetonitrile in water (both
containing 0.1% TFA) until the product eluted. After purification, the purity of all compounds
was evaluated as being more than 95% by analytical RPꢀHPLC. All fractions were lyophilized
using a FlexyꢀDry lyophilizer (FTS Systems, Warminster, PA).
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Peptide synthesis
The protected tetrapeptides BocꢀDmtꢀDꢀArg(Pbf)ꢀAbaꢀGlyꢀOH and BocꢀDmtꢀDꢀArg(Pbf)ꢀ
AbaꢀβꢀAlaꢀOH were synthesized by NαꢀFmoc solid phase methodology on 2ꢀchlorotrityl
resin, including the construction of the Abaꢀconstraint on solid phase by use of phthaloyl
protected orthoꢀformyl phenylalanine, as described in detail previously.2 After cleavage of the
fully protected peptide acid, it was coupled to the corresponding benzyl amines in solution
using DIC/HOBt. Final deprotection was performed with the mixture TFA/TES/H2O
(95/2.5/2.5) for 3 hours. The peptides were purified by preparative HPLC and characterized
by high resolution mass spectrometry (see Supporting Information). Purity was > 95% as
determined by analytical HPLC.
Functional NK1 receptor assay30
Cell line and culture conditions, aequorin charging protocol: The Chinese Hamster Ovary K1
(CHOꢀK1) cell line, stably expressing human NK1 receptor was transfected with an
apoaequorin expression vector (pER2) using Fugene6 (Roche Applied Science). Transfected
cells in the midꢀlog phase were loaded with coelenterazine and diluted 10ꢀfold as described in
detail elsewhere.2
Aequorin luminescence assay: A dilution series of peptide agonist (substance P, Sigma)
ranging from 10ꢀ11 to 10ꢀ4 M was distributed in a white 96ꢀwell plate, after which the synthetic
compounds were added to these wells to obtain the desired concentrations (ranging from 10ꢀ8
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to 10ꢀ4 M). A negative control sample (BSA medium only) was included in each row of the
96ꢀwell plate. Measurement started at the moment of injection of 50µl of the coelenterazineꢀ
loaded cell suspension, containing 2.5x 104 cells. Light emission was measured every second
for 30s after which 50µl of 10nM ATP solution (positive control) was injected.2
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Data Analysis: Luminescence data (peak integration) were calculated as described previously2
using MikroWin 2000 software (Berthold). Statistical and curveꢀfitting analyses were
performed using Prism 4.0 (GraphPad) software. Data were expressed in percentage (% RLU)
of the maximal luminescence that was detected with 10ꢀ4 M SP (without antagonist). The
competitive nature of antagonism was evaluated using the Schild plot method.22 All
antagonists analyzed in this study provided linear regression plots and were considered
competitive. The pA2 values were calculated using the Schild’s equation.22
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hNK1-CHO membrane preparation for binding assay
The hNK1ꢀCHO cells were cloned locally and grown in a humidified CO2 (5%) atmosphere at
37 °C in a Forma Scientific incubator (Thermo Forma, Series II Water Jacketed, USA) in a
Dulbecco’s DMEMꢀF12 50:50 medium (Corning Cellgro, USA) supplemented with 10%
heatꢀinactivated fetal bovine serum (Gibco, Life Technologies) and 1X PenStrep solution
(Gibco, Life Technologies, USA). Confluent cell monolayers were harvested with 1X
Dulbecco’s PBS containing 5 mM EDTA. Cells were centrifuged in a Sorvall ST 16R
centrifuge (ThermoScientific) at 1500 rpm for 3 min. The supernatants were aspirated and the
remaining pellets were placed at ꢀ80oC. On the day of the experiments, cell pellets were
thawed and homogenized with a teflonꢀglass tissue homogenizer in an iceꢀcold 50 mMTrisꢀ
HCl (pH 7.4) buffer containing 5 mM MgCl2. The membrane fractions were collected by
centrifugation in a Sorvall RC 5C Plus (ThermoScientific) at 13,000 rpm for 25 min at 4°C
and the resulting pellets were reꢀsuspended in 50mM TrisꢀHCl (pH 7.4), 5 mM MgCl2 buffer.
Protein contents of the pellet suspensions were determined by the BCA method (BioꢀRad,
BioꢀRad Laboratories, USA).
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[3H]Substance-P competition binding assay
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The test compounds were dissolved in DMSO and stored in 10 mM aliquots at ꢀ20oC. Serial
dilutions of the compounds were prepared in the binding buffer (50 mMTrisꢀHCl, pH 7.4, 5
mM MgCl2). Serial dilutions of the competitor ligands were coꢀincubated with 45 µg of
membrane protein and 0.5ꢀ0.8 nM [3H]SubstanceꢀP (36.5 Ci/mmol, PerkinElmer, USA) in a
final volume of 200 µl at RT for an hour. Total binding was determined in the absence of
competitors while nonꢀspecific binding was defined in the presence of 10 µM SubstanceꢀP
(final concentration). SubstanceꢀP (Tocris, Tocris Bioscience, UK) was also used as a control
to validate the binding assay conditions. The reactions were terminated by rapid filtration onto
UniFilter GF/B 96ꢀwell plates (PerkinElmer, USA), previously preꢀsoaked in cold 1%
polyethyleneimine solution (Sigma Aldrich, USA). Plates were washed 3 times with cold
water and dried at RT for overnight. Filter bound radioactivity was measured by a MicroBeta
2450 microplate reader (PerkinElmer, USA) using a microscintillation cocktail (PerkinElmer,
USA).
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Data analysis
Experiments were carried out in duplicate in 96ꢀwell plates, and are the average of at least
three independent experiments. The IC50 and Ki values for each test compound were
calculated by nonlinear regression and standard error of means (S.E.M) is represented. Data
analyses were performed by GraphPad Prism4 software (GraphPad, San Diego, California).
The Ki was calculated using the previously determined Kd of the [3H]SubstanceꢀP in this cell
line by saturation binding.
Functional GPI and MVD assays
The guinea pig ileum (GPI)31 and mouse vas deferens (MVD)32 bioassays were carried out as
described in detail elsewhere.33ꢀ34 A doseꢀresponse curve was determined with
[Leu5]enkephalin as standard for each ileum and vas preparation and IC50 values of the
compounds being tested were normalized according to a published procedure.35
Opioid receptor binding assays
Opioid receptor binding studies were performed as described in detail elsewhere.33 Binding
affinities for MOP and DOP receptors were determined by displacing, respectively,
[3H]DAMGO (Multiple Peptide Systems, San Diego, CA) and [3H]DSLET (Multiple Peptide
Systems) from rat brain membrane binding sites, and κ opioid receptor binding affinities were
measured by displacement of [3H]U69,593 (Amersham) from guinea pig brain membrane
binding sites. Incubations were performed for 2 h at 0 ºC with [3H]DAMGO and [3H]DSLET
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at respective concentrations of 0.72 and 0.78 nM. IC50 values were determined from logꢀdose
displacement curves, and Ki values were calculated from the IC50 values by means of the
equation of Cheng and Prusoff,36 using values of 1.3 and 2.6 nM for the dissociation constants
of [3H]DAMGO and [3H]DSLET, respectively.
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Animals
Male Albino Swiss CDꢀ1® IGS mice (30 ꢀ 35 g) obtained from Charles River Breeding
Laboratories, Germany, were housed ten mice per cage (lined with sawdust) under a standard
12/12 h light/dark cycle (lights on at 6:00 am) with food and water available ad libitum. All
experiments were performed according to the recommendations of IASP, the NIH Guide for
Care and Use of Laboratory Animals, and were approved (1214/2015) by the local Bioethics
Committee (Cracow, Poland).
Neuropathic pain model
Chronic constriction injury (CCI) model was performed according to Bennet and Xie37 and
used in our laboratory.38ꢀ39 The surgical procedure was performed under isoflurane anesthesia.
An incision was made below the mouse’s right hipbone, and the sciatic nerve was exposed.
Three ligatures with 4/0 silk thread were made around the nerve distal to the sciatic notch
with 1 mm spacing, until a brief twitch in the respective hind limb was observed. Mice were
tested for the presence of characteristics for neuropathy. All CCI mice developed allodynia
and hyperalgesia. The behavioral experiments were conducted on the 7th to 14thday following
the CCI surgical procedure.
Biochemical analysis
qRT-PCR analysis of gene expression – Spinal cord tissue for biochemical analysis was
collected on the 7th day after the CCI. Lumbar (L4ꢀL6) fragments of the spinal cord were
removed immediately after the mice were decapitated, and both the ipsilateral and
contralateral parts were dissected. The tissue samples were placed in individual tubes
containing the tissue storage reagent RNAlater (Qiangen Inc.) and stored at ꢀ70°C for RNA
isolation. Total RNA was extracted using TRIzol reagent (Invitrogen), as previously
described. RNA concentrations and quality were measured using a NanoDropNDꢀ1000
Spectrometer (NanoDrop Technologies). Reverse transcription was performed on 1 µg of total
RNA using Omniscript reverse transcriptase (Quiagen Inc.) at 37°C for 60 min. Reverse
transcription (RT) reactions were carried out in the presence of an RNase inhibitor (RNasin,
promega) and an oligo (dT16) primer (Quiagen Inc.). The cDNA was diluted 1:10 with water,
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and for each reaction, ca. 50 ng of cDNA synthesized from the total RNA of an individual
animal was used for the quantitative realꢀtime PCR (qPCR) reaction. qPCR was performed
using AssayꢀOnꢀDemand TaqMan probes according to the manufacturer’s protocol (Applied
Biosystems), and the reactions were run on an iCycler device (BioRad, Hercules). The
following TaqMan primers and probes were used: Mm00446968_m1 (Hprt, hypoxanthine
guanine phosphoribosyl transferase), Mm00436892_m1 (Tac1r, NK1 receptor),
Mm01166996_m1 (Tac1, substance P), Mm01188089_m1 (Oprm1, MOP receptor),
Mm01180757_m1 (Oprd1, DOP receptor). The expression of Hprt was measured in the
lumbar spinal cord of mice as measured on days 7 and 14 following CCI procedure and
quantified to control for variations in cDNA amounts. Hprt did not significantly change for
CCIꢀexposed mice and therefore served as an adequate housekeeping gene (data not shown).
The cycle threshold values were calculated automatically by iCycler IQ 3.0 software using
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default parameters. The RNA abundance was calculated as 2– (threshold cycle)
.
Western Blot – The ipsilateral and contralateral dorsal parts of the spinal cord (L4ꢀL6) from
naïve and CCIꢀexposed mice (7th day after injury) were collected in RIPA (Radioꢀ
Immunoprecipitation Assay) buffer with protease and phosphatase inhibitor cocktails (Sigmaꢀ
Aldrich). Tissue lysates were cleared by centrifugation (14,000× g for 30 min at 4°C) and
protein concentration in the supernatant was determined using the BCA Protein Assay Kit
(SigmaꢀAldrich). Samples containing 25 ꢁg of protein were heated in a loading buffer (4x
Laemmli Buffer, BioꢀRad) for 5 min at 98°C and then were resolved on 4–15% Criterion™
TGX™ preꢀcast polyacrylamide gels (BioꢀRad, Poland). The proteins were transferred to
ImmuneꢀBlot PVDF membranes (BioꢀRad, Poland) with semiꢀdry transfer (30 min, 25 V) and
then the membranes were blocked for 1h at RT using 5% nonꢀfat, dry milk (BioꢀRad) in Trisꢀ
buffered saline. Next, the membranes were washed in TBST (Trisꢀbuffered saline with 0.1%
Tweenꢀ20) and incubated overnight at 4 °C with the following primary antibodies: rabbit
polyclonal NK1 receptor (Novus Biologicals) 1:500; MOP receptor (Abcam) 1:500, DOP
receptor (LifeSpan) 1:500 and substance P (Santa Cruz) 1:250 or mouse polyclonal GAPDH
(Millipore) 1:5000. The membranes were then incubated for 1h in horseradish peroxidaseꢀ
conjugated antiꢀrabbit or antiꢀmouse secondary antibodies at a dilution of 1:5000. All
antibodies were diluted in the SignalBoost™ Immunoreaction Enhancer Kit (Merck Millipore
Darmstadt). The membranes were washed 4 times for 5 min each with TBST. The Clarity™
Western ECL Substrate (BioꢀRad, Poland) was used to detect immunocomplexes, which were
visualized using a Fujifilm LASꢀ4000 FluorImager system. The Fujifilm Multi Gauge
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software was used to quantify the relative levels of immunoreactivity. The immunoblots
shown are representative of 4–6 individual samples.
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Drug administration
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Three designed compounds containing both NK1 receptor antagonist and MOP and DOP
receptorꢀagonist pharmacophores: 3 (SBCHM1), 12 (RCCHM3), 13 (RCCHM6), and the
reference compounds containing only pure opioid pharmacophore: 1 (AN81) and 3 (KGOP1)
were used in the study. Drugs were dissolved in water for injections (Polpharma, Poland) and
administered intrathecally (i.t.) in 5 ꢁl dose volume through lumbar puncture between L5 and
L6 vertebrae to nonꢀanesthetized mice, according to the Hylden & Wilcox model40 described
with modifications by Fairbanks.41 The i.t. injections were performed with disposable 30
gauge 1/2ꢀinch needles (Becton Dickinson and Company, Rutherford, NJ, USA) matched to a
25ꢀꢀl syringe (Hamilton, Reno, NV, USA). Behavioral tests were performed 30, 90 and 180
min after drugs administration.
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Behavioral tests
Von Frey test: Mechanical sensitivity to nonꢀnoxious stimuli was measured by applying a set
of calibrated nylon monofilaments (0.6 – 6 g; Stoelting), in serial increments on a tested hind
paw plantar surface, until a behavioral response was observed. Responses considered as pain
behavior included paw withdrawal, shaking and licking.
Cold plate test: Sensitivity to noxious thermal stimuli was assessed with usage of Cold/Hot
Plate Analgesia Meter, Columbus Instruments. The temperature of the plate was kept at 2 °C,
and the cutꢀoff latency was 30 s. The mice were placed on the cold plate, and the time until
the hind paw was lifted was recorded.
Tail-flick test: The threshold of nociceptive pain to a thermal stimulus was assessed by tailꢀ
flick latency determined by a tailꢀflick analgesic meter (Analgesia Meter; Ugo Basile, Italy).
In this test, the beam of light was focused on the dorsal tail surface 1ꢂcm from the tip of the
tail. The baseline was determined at 2.5–4.5ꢂs. The cutꢀoff time was 9ꢂs.
RotaRod test: The motor performance of naïve mice was assessed by the RotaRod test
(Mouse RotaꢀRod, Ugo Basile, Italy). Mice were placed on a horizontal rod which was rotating
at accelerating speed, starting with 2 rpm and reaching 40 rpm within 300 seconds. The main
experiment was conducted after 3 training sessions, lasting 300 seconds each. The time was
recorded until a mouse fell off the rod. RotaRod test was conducted 30 min after drug
administration.
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ꢀ Associated Content
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An electronic supporting information file is available and contains chemistry procedures and
compound characterization.
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ꢀ Author Contributions
JS, KPB, JM and BP were in charge of performing and designing the biochemical and animal
experiments. RC, KG, AM, DT and SB have designed and prepared the hybrid opioidꢀNK1R
ligands. NNC, CL and PS carried out and supervised the functional tissue assays (GPI and
MVD). AK, JS, JVD and JVB have performed the NK1R binding and activity assays,
respectively. DT, PS, SB and BP were in charge of writing and editing the manuscript.
ꢀ Author Information
ꢀ
Corresponding Authors
Prof. Dr. Steven Ballet. Research Group of Organic Chemistry, Departments of Chemistry
and Bioꢀengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, Bꢀ1050 Brussels,
Belgium; Tel.: 0032ꢀ26293292; Fax.: 0032ꢀ26293304; eꢀmail: steven.ballet@vub.be
Prof. Barbara Przewlocka. Department of Pain Pharmacology, Institute of Pharmacology,
Polish Academy of Sciences, Smetna 12, PL 31ꢀ343, Kraków, Poland; Tel.: 0048126623398;
eꢀmail: barbara.przewlocka@gmail.com
ꢀ Acknowledgements
SB and DT thank the Research Foundation – Flanders for the financial support. The work of
JS, KPB, JM and BP was supported by MAESTRO NCN2012/06/A/NZ4/00028 grant and
statutory funds IF PAS; JS is a holder of a KNOW scholarship sponsored by the Ministry of
Science and Higher Education, Poland. The work of PWS was supported by grants from the
U.S. National Institute on Drug Abuse (NIH, DA004443) and the Canadian Institutes of
Health Research (MOPꢀ89716). Research performed by JVD and JVB was supported by the
Interuniversity Attraction Poles (IAP) program (Belgian Science Policy Grant (P7/40).
ꢀ Abbreviations
Aba: 4ꢀaminoꢀ1,2,4,5ꢀtetrahydroꢀ2ꢀbenzazepinꢀ3ꢀone; CCI: chronic constriction injury; DIC:
N,N’ꢀdiisopropylcarbodiimide, DOP: delta opioid peptide; Dmt: 2’,6’ꢀdimethyltyrosine; GPI:
guinea pig ileum; HOBt: 1ꢀhydroxybenzotriazole; MOP: mu opioid peptide; mRNA:
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messenger RNA; MPE: maximum possible effect; MVD: mouse vas deferens; NK1:
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neurokininꢀ1;
Pbf:
2,2,4,6,7ꢀpentamethyldihydrobenzofuranꢀ5ꢀsulfonyl;
qRTꢀPCR:
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quantitative realꢀtime polymerase chain reaction, GAPDH ꢀ Glyceraldehyde 3ꢀphosphate
dehydrogenase.
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Graphical abstract
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Parent compound:
Neurokinin 1 receptor antagonist
opioid agonist
n
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R1
R2
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Robust analgesic effect
in naive mice;
weak effect in CCI mice
Robust and long-lasting analgesic effect
under neuropathic pain conditions
Abolished CCI-induced hypersensitivity to:
Mechanical stimuli
CCI
Cold stimuli
Threshold stimulus
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