The Use of the Selective Imidazoline I1 Receptor Agonist Carbophenyline as a Strategy for Neuropathic Pain Relief: Preclinical Evaluation in a Mouse Model of Oxaliplatin-Induced Neurotoxicity

Article abstract of DOI:10.1007/s13311-020-00873-y

Anti-cancer therapy based on the repeated administration of oxaliplatin is limited by the development of a disabling neuropathic syndrome with detrimental effects on the patient’s quality of life. The lack of effective pharmacological approaches calls for the identification of innovative therapeutic strategies based on new targets. We focused our attention on the imidazoline I₁ receptor (I₁-R) and in particular on the selective I₁-R agonist 2-(1-([1,1′-biphenyl]-2-yl)propan-2-yl)-4,5-dihydro-1H-imidazole) (carbophenyline). The purpose of this work was the preclinical evaluation of the efficacy of carbophenyline on oxaliplatin-induced neuropathic pain in mice. Carbophenyline, acutely per os administered (0.1–10?mg?kg^?1), induced a dose-dependent anti-hyperalgesic effect that was completely blocked by the pre-treatment with the I₁-R antagonist 3 or the I₁/α₂ receptor antagonist efaroxan, confirming the I₁-R-dependent mechanism. Conversely, pre-treatment with the I₂-R antagonist BU224 did not block the anti-nociceptive effect evoked by carbophenyline. Repeated oral administrations of carbophenyline (1?mg?kg^?1) for 14?days, starting from the first day of oxaliplatin injection, counteracted the development of neuropathic pain in all behavioral tests (cold plate, Von Frey, and paw pressure tests) carried out 24?h after the last carbophenyline treatment on days 7 and 14. In the dorsal horn of the spinal cord, carbophenyline significantly decreased the oxaliplatin-induced astrocyte activation detected by immunofluorescence staining by the specific labelling with GFAP antibody. In conclusion, carbophenyline showed anti-neuropathic properties both after acute and chronic treatment with preventive effect against oxaliplatin-induced astrocyte activation in the spinal cord. Therefore, I₁-R agonists emerge as a new class of candidates for the management of oxaliplatin-induced neuropathic pain.

Full text of DOI:10.1007/s13311-020-00873-y

Neurotherapeutics
https://doi.org/10.1007/s13311-020-00873-y
ORIGINAL ARTICLE
The Use of the Selective Imidazoline I1 Receptor Agonist
Carbophenyline as a Strategy for Neuropathic Pain Relief: Preclinical
Evaluation in a Mouse Model of Oxaliplatin-Induced Neurotoxicity
Laura Micheli1 & Lorenzo Di Cesare Mannelli1 & Fabio Del Bello2 & Mario Giannella2 & Alessandro Piergentili2
Wilma Quaglia2 & Donatello Carrino3 & Alessandra Pacini3 & Carla Ghelardini1
&
#
The American Society for Experimental NeuroTherapeutics, Inc. 2020
Abstract
Anti-cancer therapy based on the repeated administration of oxaliplatin is limited by the development of a disabling neuropathic
syndrome with detrimental effects on the patient’s quality of life. The lack of effective pharmacological approaches calls for the
identification of innovative therapeutic strategies based on new targets. We focused our attention on the imidazoline I1 receptor
(I1-R) and in particular on the selective I1-R agonist 2-(1-([1,1′-biphenyl]-2-yl)propan-2-yl)-4,5-dihydro-1H-imidazole)
(carbophenyline). The purpose of this work was the preclinical evaluation of the efficacy of carbophenyline on oxaliplatin-
induced neuropathic pain in mice. Carbophenyline, acutely per os administered (0.1–10 mg kg−1), induced a dose-dependent
anti-hyperalgesic effect that was completely blocked by the pre-treatment with the I1-R antagonist 3 or the I1/α2 receptor
antagonist efaroxan, confirming the I1-R-dependent mechanism. Conversely, pre-treatment with the I2-R antagonist BU224
did not block the anti-nociceptive effect evoked by carbophenyline. Repeated oral administrations of carbophenyline
(1 mg kg−1) for 14 days, starting from the first day of oxaliplatin injection, counteracted the development of neuropathic pain
in all behavioral tests (cold plate, Von Frey, and paw pressure tests) carried out 24 h after the last carbophenyline treatment on
days 7 and 14. In the dorsal horn of the spinal cord, carbophenyline significantly decreased the oxaliplatin-induced astrocyte
activation detected by immunofluorescence staining by the specific labelling with GFAP antibody. In conclusion,
carbophenyline showed anti-neuropathic properties both after acute and chronic treatment with preventive effect against
oxaliplatin-induced astrocyte activation in the spinal cord. Therefore, I1-R agonists emerge as a new class of candidates for
the management of oxaliplatin-induced neuropathic pain.
.
.
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Key Words Imidazoline I1 receptor agonist oxaliplatin chemotherapy-induced neuropathic pain astrocytes carbophenyline
Dedicated to Prof. Maria Pigini
Introduction
Electronic supplementary material The online version of this article
Neuropathic pain is now defined by the International
Association for the Study of Pain (IASP) as “pain caused by
a lesion or disease of the somatosensory nervous system” [1].
Among different factors, nervous lesions can be induced by
neurotoxic drugs like several anticancer agents able to evoke
painful syndromes defined chemotherapy-induced neuropa-
thies. Oxaliplatin, one of the lead treatments for colorectal
cancers [2, 3], is associated with the development of a severe
persistent neuropathic pain which remains the dose-limiting
toxicity of this treatment [4]. Chronologically, oxaliplatin-
induced acute and transient hyperexcitability followed by a
chronic cumulative neuropathy experienced by almost the
50% of patients [5]. Signs and symptoms of chronic neurop-
athy include pain, paresthesia, hypoesthesia, and changes in
(https://doi.org/10.1007/s13311-020-00873-y) contains supplementary
material, which is available to authorized users.
* Lorenzo Di Cesare Mannelli
lorenzo.mannelli@unifi.it
1
Dept. of Neuroscience, Psychology, Drug Research and Child Health
- NEUROFARBA - Pharmacology and Toxicology Section,
University of Florence, Viale Gaetano Pieraccini 6,
50139 Florence, Italy
2
School of Pharmacy, Medicinal Chemistry Unit, University of
Camerino, Via S. Agostino 1, 62032 Camerino, Italy
3
Department of Experimental and Clinical Medicine, Anatomy and
Histology Section, University of Florence, Largo Brambilla 3,
50134 Florence, Italy

L. Micheli et al.
proprioception that persist during cycles [6]. In the long term,
this syndrome has a deleterious impact on cancer survivors,
often being associated with sleep disturbance, depressive
symptoms, and impaired health-related quality of life [7].
Several molecular mechanisms participate to oxaliplatin neu-
rotoxicity involving early targets in the peripheral nervous
system as well as direct and indirect alterations of the neuronal
and glial compartments of the central nervous system [8–13].
No treatments are currently available to reduce oxaliplatin-
dependent neuropathic pain, further pain-relieving drugs used
against neuropathic pain are not satisfactory due to limited
efficacy and side effects [14, 15]. For these reasons, efforts
are necessary to explore novel molecular targets for the phar-
macological management of neuropathic pain.
Recent studies have highlighted that the imidazoline I2 re-
ceptor (I2-R) activation by selective ligands such as 2-BFI [16]
or phenyzoline (1) (Fig. 1) [17], alone or in combination with
opioids, triggers a significant anti-hyperalgesic effect.
Therefore, this receptor system has been suggested as a prom-
ising target for the management of chronic pain [18–20]. The
I2-R is a member of a wider family of imidazoline receptors,
also including I1-R and I3-R, which play important roles in
central cardiovascular regulation and in insulin secretion, re-
spectively [21, 22]. In particular, I1-R is involved in the hy-
potensive activity of clonidine and related compounds
supporting the idea that the I1-Rs are upstream from the α2-
adrenergic receptors and work in tandem for its effect on
blood pressure [23]. Additionally, I1-R activation may sup-
press sympathetic outflow at postganglionic sympathetic neu-
rons through modulation of several signal transduction sys-
tems including N-type calcium channels, G protein inwardly
rectifying potassium channel, adenosine receptors,
phosphatidyl-choline-specific phospholipase C, and nicotinic
receptors [24–29]. On this basis, a possible role of I1-R in pain
modulation can be theoretically suggested although so far un-
explored. In the present work we studied the effects of I1-R
modulation against oxaliplatin-induced neuropathic pain by
using the selective I1-R agonist compound 2 (in this paper
named carbophenyline, highly selective over I2, α2, and 5-
HT1A receptors [30, 31]) (Fig. 1). Its effects on pain threshold
were evaluated after single or repeated administrations in the
absence or presence of the I1-R antagonist 3 [2-(1-methyl-2-
phenylethyl)-4,5-dihydro-1H-imidazole] (Fig. 1) [32], highly
selective over I2, α2 and 5-HT1A receptor and the well-known
I1/α2 receptor antagonist efaroxan [33] which proved to be
selective over several other biological targets [33; https://
pubchem.ncbi.nlm.nih.gov/compound/Efaroxan#section=
BioAssay-Results]. Moreover, the I2 antagonist BU224 was
also used to exclude the involvement of I2-R [34]. Finally, to
analyze the impact of I1-R stimulation on the maladaptive
plasticity of the neuropathic central nervous system, the
effect of carbophenyline on oxaliplatin-induced astrocyte ac-
tivation in the dorsal horn of the spinal cord was assessed.
Materials and Methods
Drugs
The I1-R agonist 2-(1-([1,1′-biphenyl]-2-yl)propan-2-yl)-4,5-
dihydro-1H-imidazole) (2, carbophenyline) and the I1-R an-
tagonist 2-(1-phenylpropan-2-yl)-4,5-dihydro-1H-imidazole
(3) were obtained treating methyl 3-([1,1′-biphenyl]-2-yl)-2-
methylpropanoate and methyl 2-methyl-3-phenylpropanoate,
respectively, with ethylenediamine and trimethyl aluminum at
110 °C in toluene [30, 32]. Efaroxan hydrochloride and
BU224 hydrochloride were purchased by Tocris. Oxaliplatin
Fig. 1 Chemical structures of compounds 1–4, sharing a common bioversatile scaffold

The Use of the Selective Imidazoline I1 Receptor Agonist Carbophenyline as a Strategy for Neuropathic...
and pregabalin were purchased by Carbosynth (Compton,
UK).
Oxaliplatin-Induced Neuropathic Pain
2.4 mg kg−1 oxaliplatin was dissolved in 5% glucose solution
and administered intraperitoneally (i.p.) for 5 consecutive
days every week for 2 weeks (10 i.p. injections) [36 with
minor modification, 37]. Control animals received an equiva-
lent volume of 5% glucose i.p. (vehicle).
Cell Cultures
The HT-29 human colon cancer cell line were acquired from
the American Type Culture Collection. HT-29 were cultured
in Complete Medium (CM) composed by high-glucose
DMEM with 10% FBS, 2 mM L-glutamine, 100 IU/mL pen-
icillin, and 100 μg mL−1 streptomycin (Sigma-Aldrich, Italy).
Compound Administration and Study of the
Pharmacodynamic Mechanisms
Cell Viability Assay
When neuropathic pain was fully established (starting from
day 14), carbophenyline (0.1–10 mg kg−1) was suspended in
1% solution of carboxymethylcellulose sodium salt (CMC)
and per os (p.o.) acutely administered ranging from
0.1 mg kg−1 to 10 mg kg−1 to evaluate its symptomatic effi-
cacy. Pregabalin (30 mg kg−1, p.o.) was used as reference drug
[38]. Afterwards, to highlight a preventive effect, repeated per
os administrations of 1 mg kg−1 of carbophenyline were car-
ried out daily from the beginning of oxaliplatin administration
(day 1) to the end of the experiment (day 14). Control animals
were treated with vehicles (5% glucose and CMC 1%).
For the study of the pharmacodynamic mechanisms, the
selective I1-R antagonist 3, I1/α2 receptor antagonist efaroxan
and selective I2-R antagonist BU224 were used. All antago-
nists were suspended in CMC 1% and p.o. administered
15 min before carbophenyline. For 3 and BU224 antagonists
we used the first dose non hyperalgesic as shown in
Supplementary table 1. Control animal were treated with ve-
hicles (5% glucose and CMC 1%).
To plate HT-29 cells (1 × 104 cells/well), 96-well cell culture
plates (Corning) were used. After 48 h, the experiments were
performed. Cells were incubated in serum-free DMEM with
0.3–100 μM oxaliplatin (Carbosynth, Compton, Berkshire,
UK) in the absence or presence of carbophenyline 10 μM
for 48 h. Cell viability was evaluated by the reduction of
3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT; Sigma-Aldrich, Italy) as an index of mitochon-
drial compartment functionality. After 48 h incubation,
1 mg mL-1 MTT in serum-free DMEM without phenol red
was added into each well and incubated for 30 min at 37 °C.
After washing the formazan crystals were dissolved in 150 μL
dimethyl sulfoxide. The absorbance was measured at 550 nm.
Experiments were performed in triplicate.
Animals
CD-1 mice (Envigo, Varese, Italy) weighing 20–25 g at the
beginning of the experimental procedure were used. Animals
were housed in the Centro Stabulazione Animali da
Laboratorio (University of Florence) and used at least 1 week
after their arrival. Ten mice were housed per cage (size
26 cm × 41 cm); animals were fed a standard laboratory diet
and tap water ad libitum and kept at 23 1 °C with a 12 h
light/dark cycle (light at 7 A.M.). All animal manipulations
were carried out according to the Directive 2010/63/EU of the
European parliament and of the European Union council (22
September 2010) on the protection of animals used for scien-
tific purposes. The ethical policy of the University of Florence
complies with the Guide for the Care and Use of Laboratory
Animals of the US National Institutes of Health (NIH
Publication No. 85–23, revised 1996; University of Florence
assurance number: A5278–01). Formal approval to conduct
the experiments described was obtained from the Italian
Ministry of Health (No. 54/2014-B) and from the Animal
Subjects Review Board of the University of Florence.
Experiments involving animals have been reported according
to ARRIVE guidelines [35]. All efforts were made to mini-
mize animal suffering and to reduce the number of animals
used.
Paw Pressure Test
Mechanical hyperalgesia was determined by measuring the
latency in seconds to withdraw the paw away from a constant
mechanical pressure exerted onto the dorsal surface [39]. A
15 g calibrated glass cylindrical rod (diameter = 10 mm)
chamfered to a conical point (diameter = 3 mm) was used to
exert the mechanical force. The weight was suspended verti-
cally between two rings attached to a stand and was free to
move vertically. A single measure was made per animal. A
cutoff time of 40 s was used.
Cold Plate
Thermal allodynia was assessed using the cold plate test. With
minimal animal–handler interaction, mice were taken from
home-cages, and placed onto the surface of the cold plate
(Ugo Basile, Varese, Italy) maintained at a constant tempera-
ture of 4 °C 1 °C. Ambulation was restricted by a cylindrical
Plexiglas chamber (diameter, 10 cm; height, 15 cm), with
open top. A timer controlled by foot peddle began timing
response latency from the moment the mouse was placed onto

L. Micheli et al.
the cold plate. Pain-related behavior (licking of the hind paw)
was observed, and the time (seconds) of the first sign was
recorded. The cutoff time of the latency of paw lifting or
licking was set at 30 s [40].
Mannheim, Germany). Microglia and astrocyte morphology
were assessed by inspection of at least three fields (× 40
0.75NA objective) in the dorsal horn.
Quantitative analysis of GFAP and Iba1-positive cells was
performed by collecting at least three independent fields
through a × 20 0.5NA objective. Immunofluorescent staining
was measured as mean fluorescence intensity using ImageJ
software (ImageJ, National Institute of Health, USA, https://
imagej.nih.gov/) by automatic thresholding algorithm.
Furthermore, GFAP or Iba-1 -positive cells were quantified
by thresholding automatic count. Results, given as mean fluo-
rescent intensity (arbitrary units) by the thresholded fluores-
cent signal, revealed a common trend between GFAP/Iba-1
expression and astrocyte/microglial cell number (data not
shown). Five spinal cord sections were analyzed for each
animal.
Von Frey
The animals were placed in 20 × 20 cm Plexiglas boxes
equipped with a metallic meshy floor, 20 cm above the bench.
A habituation of 15 min was allowed before the test. An elec-
tronic Von Frey hair unit (Ugo Basile, Varese, Italy) was used:
the withdrawal threshold was evaluated by applying force
ranging from 0 to 5 g with 0.2 g accuracy. Punctuate stimulus
was delivered to the mid-plantar area of each anterior paw
from below the meshy floor through a plastic tip and the
withdrawal threshold was automatically displayed on the
screen. Paw sensitivity threshold was defined as the minimum
pressure required to elicit a robust and immediate withdrawal
reflex of the paw. Voluntary movements associated with lo-
comotion were not taken as a withdrawal response. Stimuli
were applied on each anterior paw with an interval of 5 s. The
measure was repeated 5 times and the final value was obtained
by averaging the 5 measures [41, 42].
Statistical Analysis
Behavioral measurements were performed on ten mice for
each treatment carried out in 2 different experimental sets.
All assessments were made by researchers blinded to animal
treatments. Results were expressed as mean (S.E.M.) with
one-way analysis of variance. A Bonferroni’s significant dif-
ference procedure was used as a post hoc comparison. P
values < 0.05 or < 0.01 were considered significant. Data were
analyzed using the Origin 9 software (OriginLab,
Northampton, MA, USA).
Immunohistochemistry
On day 14, after behavioral measurements, mice were
sacrificed and the lumbar spinal cord segments were removed,
postfixed in 4% paraformaldehyde, and then cryoprotected in
30% sucrose solution at 4 °C. Slide-mounted cryostat sections
(5 μm) were processed for indirect immunofluorescence
histochemistry.
Results
Formalin-fixed cryostat sections (5 μm) were incubated for
30 min in ready-to-go blocking solution (Bio-Optica, Milan,
Italy) at room temperature to block unspecific binding. The
primary antibodies, overnight incubated at 4 °C, were directed
against Iba1 (rabbit, 1:200; Wako Chemicals, Richmond,
USA) for microglial staining or against glial fibrillary acidic
protein (GFAP; rabbit, 1:200; Dako, USA) for astrocyte stain-
ing. After rinsing in PBST, sections were incubated in donkey
anti-rabbit IgG secondary antibody labelled with Alexa Fluor
488 (1:500, Invitrogen, Milan, Italy) at room temperature for
1 h. Nuclei were stained with DAPI (4′,6-diamidin-2-
fenilindolo; 1:2000; Invitrogen, Milan, Italy).
Negative control sections (no exposure to the primary
antisera) were processed concurrently with the other sections
for all immunohistochemical studies. We obtained a single
optical density value for the dorsal horns by averaging the
two sides in each mouse, and these values were compared to
the homologous average values from the vehicle-treated
animals.
Behavioral Evaluations
The effects of carbophenyline against neuropathic pain were
studied in a mouse model of chemotherapy-induced neuro-
pathic pain. The neurotoxicity of oxaliplatin was used to
evoke a neuropathic syndrome characterized by thermal and
mechanical hypersensitivity and allodynia after repeated ad-
ministrations (2.4 mg kg−1, 10 intraperitoneal injections)
(Fig. 2). On day 14, the response to a cold non-noxious stim-
ulus (allodynia-like symptom) was measured by the cold plate
test. The pain threshold of oxaliplatin-treated animals de-
creased to 9.8 0.2 s in comparison to 18.2 0.4 s of the
control group (vehicle + vehicle-treated animals). Increasing
doses of carbophenyline (0.1–10 mg kg−1), acutely per os
administered, reduced thermal allodynia in a dose-dependent
manner. The single administration of compound at
10 mg kg−1 was able to fully counteract oxaliplatin-induced
neuropathic pain 30 min after injection with the onset 15 min
after treatment that lasted up to 45 min. A dose of 1 mg kg−1
increased the pain threshold of the animals starting 15 min
after treatment with a peak at 45 min. The anti-
Images were acquired by a motorized Leica DM6000B
microscope equipped with a DFC350FX camera (Leica,

The Use of the Selective Imidazoline I1 Receptor Agonist Carbophenyline as a Strategy for Neuropathic...
Fig. 2 Effect of acute carbophenyline administrations on pain behavior
induced by oxaliplatin. Pain threshold to (a) a non-noxious thermal stim-
ulus (cold plate test), (b) to a noxious mechanical stimulus (paw pressure
test) and (c) to a non-noxious mechanical stimulus (Von Frey test) were
measured. Oxaliplatin (2.4 mg kg−1, i.p.) was administered for 2 weeks
(10 injections). Starting from day 14, carbophenyline was acutely per os
administered (0.1–10 mg kg−1) and measurements assessed before and
15, 30, 45, 60 min after injection. Control animals were treated with
vehicles. Each value represents the mean S.E.M. of 10 animals per
group performed in 2 experimental sets. Statistical analysis is one-way
ANOVA followed by Bonferroni’s post hoc comparison. ^^P < 0.01 vs
vehicle + vehicle; **P < 0.01 vs oxaliplatin + vehicle
hypersensitivity effect vanished at 60 min. The lowest dose
(0.1 mg kg−1) was inactive. Pregabalin was used as reference
drug. The administration of the drug at the dose of 30 mg kg−1
significantly increased the pain threshold 30 min after treat-
ment with the effect that remained active up 45 min without
completely counteract oxaliplatin-induced thermal allodynia
(Fig. 2a). The acute anti-hyperalgesic and anti-allodynic ef-
fects of carbophenyline were also evaluated by paw pressure
test and Von Frey test, respectively (Fig. 2 b and c). Results
obtained were comparable to that achieved by the cold plate
test. Carbophenyline evoked a dose-dependent anti-neuro-
pathic effect that reached the higher effect with the dose of
10 mg kg−1, 30 min after the administration.
To confirm the pharmacodynamics of carbophenyline,
three specific antagonists were used: 3 (selective I1-R antago-
nist), efaroxan (I/α2 receptor antagonist), and BU224 (selec-
tive I2-R antagonist). As shown in Fig. 3, the pre-treatment
(15 min before carbophenyline administration) with 3 10
(mg kg−1, p.o.) was able to completely abolish the pain-
relieving effect of carbophenyline till the end of the
experiment. The same result was obtained with the pre-
treatment with efaroxan (10 mg kg−1, p.o., 15 min before
carbophenyline administration). BU224 (3 mg kg−1 p.o.,
15 min before carbophenyline administration) was not able
to modify the efficacy of carbophenyline over the time of
observation (Fig. 3).
Repeated treatments of carbophenyline (1 mg kg−1 p.o.)
were performed to evaluate the preventive effect of the com-
pound in the same model of oxaliplatin-induced neuropathic
pain. Oxaliplatin-treated animals were daily injected with
carbophenyline starting from the same day of antitumor ad-
ministration. The response to a thermal (cold plate test) and
mechanical (paw pressure and Von Frey tests) stimuli was
measured on days 7 and 14, 24 h after the last treatment
(Fig. 4). Oxaliplatin-induced neuropathic pain increased dur-
ing time reaching the plateau on week 2 (day 14) as shown by
Fig. 4. Repeated daily administration of carbophenyline in-
creased the pain threshold of oxaliplatin-treated animals at
all time points without development of tolerance to the anti-
hypersensitivity effect. The effect evoked by carbophenyline

L. Micheli et al.
HT-29 Cell Line
Aimed to evaluate a possible interaction between the treat-
ment with carbophenyline and the therapeutic properties of
oxaliplatin, we analyzed the vitality of the human colorectal
cancer cell line HT-29. Table 1 shows oxaliplatin lethality
after 48 h incubation (0.3–100 μM) in the absence or presence
of carbophenyline. Ten μM carbophenyline did not signifi-
cantly alter the oxaliplatin-dependent decrease of cell viabili-
ty. Carbophenyline per se did not influence HT-29 viability
up to 100 μM as shown in Table 2.
Discussion
Fig. 3 Study of carbophenyline pharmacodynamic profile. Pain was
induced by repeated treatment with oxaliplatin. The hypersensitivity to
a cold stimulus was measured by the cold plate test. Carbophenyline was
administered per os at 10 mg kg−1. The I1 receptor antagonist 3
(10 mg kg−1), the I1/α2 receptor antagonist efaroxan (10 mg kg−1) and
the I2 receptor antagonist BU224 (3 mg kg−1) were p.o. administered
15 min before carbophenyline; measurements were assessed before and
15, 30, 45, 60 min after carbophenyline injection. Control animals were
treated with vehicles. Each value represents the mean S.E.M. of 10
animals per group performed in 2 experimental sets. Statistical analysis
is one-way ANOVA followed by Bonferroni’s post hoc comparison.
^^P < 0.01 vs vehicle + vehicle; **P < 0.01 vs oxaliplatin + vehicle;
°°P < 0.01 vs oxaliplatin + carbophenyline
The present data show for the first time the involvement of the
I1-R in the signaling of neuropathic pain, highlighting the
properties of the selective I1-R agonist carbophenyline in re-
lieving pain alterations in a mouse model of oxaliplatin-
induced neurotoxicity. Moreover, repeated administration of
carbophenyline prevented the maladaptive changes that occur
to the astrocyte population in the central nervous system. A
new class of potential neuropathic pain relievers has been
outlined.
Many different pharmacological actions have been associ-
ated with imidazoline compounds [21] and our research on
them has highlighted that the 2-substituted imidazoline ring
linked to an aromatic moiety (Ar) by a biatomic bridge (X) is a
bioversatile scaffold to obtain agents interacting with different
targets [43–46] (Fig. 1). Some of these imidazoline ligands
demonstrated to be efficacious as anti-neuropathic agents. In
particular, in addition to the already mentioned I2 ligand
phenyzoline (1), characterized by a –CH2CH2- bridge, the
dual α2/5-HT1A receptor agonist S-(-)-biphenyline (4) [47,
48], bearing an –OCH(CH3)- bridge and an ortho phenyl sub-
stituent in the aromatic ring, showed a significant pain-
relieving effect in a rat model of neuropathic pain [49]. The
isosteric substitution of the oxygen atom of the bridge with a
CH2 group produced derivative carbophenyline, characterized
by an interesting modulation of the biological profile. Indeed,
carbophenyline behaves as an I1-R agonist with high selectiv-
ity over I2, α2, and 5-HT1A receptors [30, 31, 48]. The profit-
able activation of I1-R makes carbophenyline a potent anti-
hypertensive agent [30, 31].
remained stable on days 7 and 14 as highlighted by the cold
plate and paw pressure tests (Fig. 4 a and b, respectively)
counteracting oxaliplatin-induced neuropathic pain. Similar
results were obtained measuring the response to a mechanical
non-noxious stimulus (Von Frey test, Fig. 4c), carbophenyline
increased its efficacy during time completely abolishing me-
chanical allodynia on day 14.
Analysis of Glial Cells in the Spinal Cord
The central nervous system was analyzed to assess the
capability of carbophenyline to intervene against the mal-
adaptive plasticity induced by oxaliplatin treatment. In the
spinal cord, repeated oxaliplatin injections induced astro-
cyte activation, measured as increased GFAP fluorescence
intensity (day 14) on the entire surface and, particularly,
in the superficial laminae (Fig. 5b) with respect to the
control animals (Fig. 5a). The repeated administration of
carbophenyline significantly prevented the astrocytic re-
sponse to oxaliplatin as shown in Fig. 5c. As previously
reported [9], spinal microglia was not altered by
oxaliplatin on day 14 (Supplementary Figure S1); the
co-treatment with carbophenyline did not modify this con-
dition (Fig. S1).
Although imidazoline binding sites attracted attention in
nociception, only the I2-R was studied. A possible role of I1-
R can be hypothesized analyzing the efficacy of clonidine (a
molecule possessing I1-R affinity in addition to the well-
known adrenergic α2-mediated effects) against neuropathic
and cancer pain [50–53], even if a possible I1-R component
in clonidine anti-nociceptive effects was not described. Stone
et al. [54] showed that there is a possible synergy between I1-R
and opioid receptors at the spinal level based on the use of the

The Use of the Selective Imidazoline I1 Receptor Agonist Carbophenyline as a Strategy for Neuropathic...
Fig. 4 Effects of repeated administration of carbophenyline against pain
induced by oxaliplatin. Pain threshold to a non-noxious thermal stimulus
as measured by the cold plate test (a). Sensitivity to a noxious mechanical
stimulus as measured by the paw pressure test (b). Pain threshold to a
non-noxious mechanical stimulus as measured by the Von Frey test (c).
Behavioral tests were performed on days 7 and 14 after the beginning of
oxaliplatin and carbophenyline administrations, 24 h after the last
treatment. Oxaliplatin (2.4 mg kg−1, i.p.) was administered for 2 weeks
(10 injections) whereas carbophenyline (1 mg kg−1, p.o.) was daily ad-
ministered, starting from day 1 of oxaliplatin injection. Control animals
were treated with vehicles. Each value represents the mean S.E.M. of 10
animals per group performed in 2 experimental sets. Statistical analysis is
one-way ANOVA followed by Bonferroni’s post hoc comparison.
^^P < 0.01 vs vehicle + vehicle; **P < 0.01 vs oxaliplatin + vehicle
selective I1-R agonist, diethyl-phenyl-amino-imidazoline,
ST91. Exploring the possible anatomical and molecular im-
pact of I1-R in the nervous system for justifying the pain-
relieving effect, some interesting data emerge. I1 binding sites
were found in bovine, rabbit, rat, and human brainstem and
brain [55]. In the brainstem, I1-Rs were detected predominant-
ly on non-mitochondrial membranes [56]; in the brain, they
are particularly expressed by synaptic membranes, and most
likely from presynaptic terminals [55]. Among molecular
mechanisms mediated by I1-R, Edwards and Ernsberger [57]
showed that the receptor activation can abolish the nerve
growth factor–activated signalling pathway by increasing the
levels of a specific phosphatase through ERK dephosphoryla-
tion. This effect could be particularly relevant in pain modu-
lation since the nerve growth factor (as others growth factors)
is a potent pain inducer [58] that seems unable to separate the
neuroprotective effect from the algic one probably following
the evolutionary positive alarm role of physiological pain.
Moreover, moxonidine, an anti-hypertensive drug, inhibited
voltage-dependent N-type Ca2+ current in rat ganglion neu-
rons via activating I1-R attenuating repetitive firing [29].
Neuronal N-type Ca2+ current is closely related to nociception
and the pivotal role of its modulation in the pharmacodynamic
of anti-neuropathic drugs was recently described by our group
[59]. On the contrary, other authors correlated the I1-R activa-
tion in the striatum with pro-excitatory signals [60] that cannot
fit with pain relief indicating that the nervous I1-R signaling is
complex and dependent on the anatomical localization.
Furthermore, a few data indicate a I1-R role in neuropro-
tection. The stimulation of this receptor participates in the
improvement of locomotor recovery in mice subjected to spi-
nal cord injury treated with agmatine [61]. Treatment with
moxonidine significantly attenuated locomotor activity, grip
strength, learning, and memory impairments in a model of
Huntington’s disease [62].
Considering the wide expression of I1-R in several areas of
the CNS [55] and that no studies reported on the anti-
neuropathic efficacy of selective I1-R ligands, we thought it

L. Micheli et al.
Fig. 5 Astrocytic profile in the spinal cord. The effect of repeated
treatment with carbophenyline (1 mg kg−1) was evaluated in
oxaliplatin-treated mice on day 14. The number of GFAP-positive cells
was measured in the dorsal horn of the L4–L5 spinal cord. Transverse
sections of spinal cord imaged with × 20 objective (scale bar = 50 μm);
insert shows morphological characteristics of a representative astrocytic
cell. Histograms show the quantitative analysis of GFAP fluorescence
intensity. Each value represents the mean of 6 mice, performed by ana-
lyzing 4 independent fields for both sides of the dorsal horn of the lumbar
spinal cord. Statistical analysis is one-way ANOVA followed by
Bonferroni’s post hoc comparison. ^^P < 0.05 vs vehicle + vehicle;
**P < 0.01 vs oxaliplatin + vehicle
Table 1 HT-29 cell viability after oxaliplatin alone or in combination
with carbophenyline 48 h incubation
Table 2 HT-29 cell
viability after
carbophenyline 48 h
incubation
Cell viability %
48 h incubation
Cell viability %
48-h incubation
Carbophenyline
(μM)
0
100.0 1.9
98.0 1.4
96.3 3.3
97.2 1.9
94.2 1.4
41.7 1.9***
Oxaliplatin
Control
Carbophenyline
1
concentration (μM)
10 μΜ
10
30
100
300
0
0.3
1
100.0 1.3
91.8 1.7
89.0 2.2
79.7 1.4
72.5 1.1
64.5 1.2
31.8 0.3
98.2 2.2
91.1 2.4
91.6 1.1
79.3 0.9
68.2 0.7
63.4 0.9
31.4 1.7
3
10
30
100
HT-29 cells (cells/well) were treated with
i n c r e a s i n g c o n c e n t r a t i o n s o f
carbophenyline (1–300 μM) for 48 h.
Cell viability was measured by the MTT
assay. The control condition was arbitrari-
ly set as 100% and values are expressed as
the mean S.E.M. of three experiments. t
test was used to compare the treated sam-
ples to the control. ***P < 0.001 vs the
control condition
HT-29 cells (cells/well) were treated with increasing concentrations of
oxaliplatin (1–100 μM) in the presence or absence of 10 μΜ
carbophenyline. Incubation was allowed for 48 h. Cell viability was mea-
sured by the MTT assay. The control condition was arbitrarily set as
100% and values are expressed as the mean S.E.M. of three experi-
ments. t test was used to compare carbophenyline treated samples in
combination with oxaliplatin to the oxaliplatin-treated samples

The Use of the Selective Imidazoline I1 Receptor Agonist Carbophenyline as a Strategy for Neuropathic...
interesting to investigate the role played by I1-R in the
oxaliplatin-induced neuropathic pain using the selective I1-R
agonist carbophenyline. The selection of carbophenyline is
rationally justified on the basis of its structural analogy with
other anti-neuropathic imidazoline compounds at our dispos-
al, in particular derivative 4 showing a significant pain-
relieving effect in a rat model of neuropathic pain [49].
In vivo experiments demonstrated the symptomatic proper-
ties of carbophenyline in relieving oxaliplatin-induced
allodynia and hyperalgesia after a single acute injection. The
relevance of the I1-R in the pain-relieving pharmacodynamic of
carbophenyline has been validated through the use of 3,
efaroxan, and BU224 (I1-, I1/α2-, and I2-R antagonists, respec-
tively) demonstrating the selective involvement of type I1-R.
Moreover, carbophenyline prevented the development of
oxaliplatin-induced neuropathic pain after a repeated adminis-
tration, and the effect remained stable both 7 and 14 days over
treatment. The described preventive profile allows us to exclude
the development of tolerance to the anti-hypersensitivity ef-
fects. Further, it suggests possible neuroprotective properties
of carbophenyline able to modify the nervous system alterations
leading the establishment and persistence of neuropathic pain.
This aspect was studied in the present research analyzing
the possible impact of I1-R stimulation on spinal cord alter-
ations evoked by oxaliplatin. The repeated treatment with
carbophenyline prevented the activation of astrocytes in the
dorsal horns. Glia cells contribute to the persistence of pain, an
effect that in neuropathic conditions prevails on the usual ho-
meostatic functions [63]. The metabolic activation of microg-
lia and astrocytes participates in the maladaptive plasticity of
the central nervous system, facilitating nociceptive processes,
generating clinical pain hypersensitivity, and making neuro-
pathic pain an autonomous disease state [64]. Despite the lim-
ited ability of oxaliplatin to cross the blood–brain barrier [65],
in our previous study we demonstrated that microglia and
astrocytes cooperated to promote the initial phase of spinal
cord sensitization but, although microglia conclude the task
in the early phase of oxaliplatin administration, astrocytes
maintain their activation contributing to the pain persistence
[10]. These data are supported by the information that the
spinal intrathecal infusion of fluorocitrate, an astrocyte selec-
tive inhibitor, fully prevented oxaliplatin-evoked pain alter-
ations [10]. The present results revealed the modulatory role
of carbophenyline on oxaliplatin-induced spinal astrocyte ac-
tivation suggesting its ability to protect from the maladaptive
plasticity of the central nervous system, a mechanism that
could offer an explanation for the prevention of pain develop-
ment demonstrated after a repeated treatment. The present first
demonstration regarding the potential of I1-R agonists in pain
relief adds a fragment to the very limited information regard-
ing the role of this receptor in nervous signals.
of possible interactions with the anticancer effect of the
drug as the primary treatment outcome. As a preliminary
evaluation we analyzed the effect of carbophenyline on
oxaliplatin lethality against the human colon adenocarci-
noma cell line HT-29, representing a main clinical target
for this platin derivative [2, 3]. No interferences with the
concentration-dependent effect of oxaliplatin were regis-
tered after 48 h treatment with 10 μM of carbophenyline,
a concentration about 65-fold higher in comparison to the
I1-R affinity [30, 31]. Carbophenyline per se did not alter
HT-29 cell viability suggesting the lack of a direct effect
against cancer cells. On the base of pharmacophore
modelling and virtual docking study, I1-R agonists were
suggested as possible apoptotic agents in cancer cells
[66]. In the pheochromocytoma-derived cell line PC12,
not specific I1-R ligands showed antiproliferative effect
[55]. Generally, imidazolines can sensitize cancer cells
to antineoplastic DNA damaging agents [67]. The role
of I1-R in cancer and the effects of specific ligands, like
carbophenyline, in cancer mechanisms as well as in the
possible pharmacological interactions with antitumor
drugs deserve deep investigation.
The development of oxaliplatin neuropathy is a clinical
issue in patients receiving a cumulative dose exceed 780–
850 mg/m2, approximately two-thirds will have symptoms
1 year after treatment or beyond [4, 68]. The lack of effective
therapies claims for new pharmacological approaches: in this
context I1-R stimulation rises to a very interesting potential.
In conclusion, for the first time the I1-R has been demon-
strated to be a valuable and effective target for the manage-
ment of oxaliplatin-induced neuropathic pain. The potent and
selective I1-R agonist carbophenyline is a lead compound for
the development of an innovative class of pain killers. The
novelty of the obtained results stimulates the continuation of
the research both from chemical and biological point of view.
In fact, the presence in carbophenyline of an asymmetric car-
bon atom suggests the examination of the influence of stereo-
chemistry on its anti-neuropathic effect. Moreover, it might be
of interest to investigate whether I1-R ligands can be active
also in other type of neuropathic pain as well as to highlight
the molecular pharmacodynamic of pain relief.
Acknowledgments This research was funded by the Italian Ministry of
Instruction, University and Research (MIUR), by the University of
Florence and by the University of Camerino (Fondo di Ateneo per la
Ricerca 2018).
Required Author Forms Disclosure forms provided by the authors are
available with the online version of this article.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
A crucial point in the study of novel treatments against
chemotherapy-induced neuropathic pain is the evaluation

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