Synergic stimulation of serotonin 5-HT1A receptor and α2-adrenoceptors for neuropathic pain relief: Preclinical effects of 2-substituted imidazoline derivatives

Article abstract of DOI:10.1016/j.ejphar.2017.06.023

Neuropathic pain affects millions of people causing disability and impairing quality of life. Commonly used analgesics are generally characterized by limited therapeutic outcomes. The serotonin 5‐HT_1A receptor and the α₂ adrenergic receptors are involved in central nociceptive mechanisms with a pivotal role in the inhibitory descending pain pathway. Since their stimulation may modulate the nervous signaling altered by neuropathies, the purpose of the present research is the study of the combined activation of 5‐HT_1A and α₂ receptors by rationally designed imidazoline ligands ((S)-(-)-1 and 2–5) in a rat model of neuropathic pain (chronic constriction injury - CCI). On day 14 after nerve damage, the acute administration per os (p.o.) of low doses of (S)-(-)-1 (0.1–1?mg/kg) was able to significantly increase the pain threshold to mechanical noxious stimuli for more than 1?h. (S)-(-)-1 efficacy was confirmed by the decrease of spontaneous pain evaluated as hind limb weight bearing alterations. The clinically-used compound gabapentin (100?mg/kg p.o.) induced a pain relieving effect similar to (S)-(-)-1 administered at 100 fold lower dose. In the same model, the selected analogues, compounds 2–5 (1?mg/kg p.o.) were effective 30?min after administration. In particular, 5 fully reverted the CCI-induced hypersensitivity. The pain relieving activity of 5 was significantly prevented by the selective 5-HT_1A receptor antagonist WAY 100635 (1?mg/kg intraperitoneally, i.p.) and, at a lesser extent, by the α₂ antagonist yohimbine (3?mg/kg i.p.). A novel pharmacodynamic approach to the treatment of neuropathic pain is presented.

Full text of DOI:10.1016/j.ejphar.2017.06.023

Author’s Accepted Manuscript
Synergic stimulation of serotonin 5-HT receptor
1A
and α -adrenoceptors for neuropathic pain relief:
2
preclinical effects of 2-substituted imidazoline
derivatives
Lorenzo Di Cesare Mannelli, Carla Ghelardini,
Laura Micheli, Fabio Del Bello, Mario Giannella,
Alessandro Piergentili, Wilma Quaglia
www.elsevier.com/locate/ejphar
PII:
S0014-2999(17)30431-4
http://dx.doi.org/10.1016/j.ejphar.2017.06.023
EJP71277
DOI:
Reference:
To appear in:
European Journal of Pharmacology
Received date: 16 May 2017
Revised date: 16 June 2017
Accepted date: 16 June 2017
Cite this article as: Lorenzo Di Cesare Mannelli, Carla Ghelardini, Laura
Micheli, Fabio Del Bello, Mario Giannella, Alessandro Piergentili and Wilma
Quaglia, Synergic stimulation of serotonin 5-HT
receptor and α
1A
2
adrenoceptors for neuropathic pain relief: preclinical effects of 2-substituted
imidazoline derivatives, European Journal of Pharmacology
http://dx.doi.org/10.1016/j.ejphar.2017.06.023
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Synergic stimulation of serotonin 5-HT1A receptor and 2-adrenoceptors for neuropathic pain
relief: preclinical effects of 2-substituted imidazoline derivatives
Lorenzo Di Cesare Mannelli1*, Carla Ghelardini1, Laura Micheli1, Fabio Del Bello2, Mario
Giannella2, Alessandro Piergentili2, Wilma Quaglia2
1Department of Neuroscience, Psychology, Drug Research and Child Health - Neurofarba -
Pharmacology and Toxicology Section, University of Florence, Viale Pieraccini 6, 50039 Firenze,
Italy
2School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Via S. Agostino 1, 62032
Camerino, Italy
*Correspondence to: Dept. of Neuroscience, Psychology, Drug Research and Child Health -
Neurofarba - Pharmacology and Toxicology Section, University of Florence, Viale Pieraccini 6,
50139, Florence, Italy. Tel.: +00390552758395. lorenzo.mannelli@unifi.it
Abstract
Neuropathic pain affects millions of people causing disability and impairing quality of life.
Commonly used analgesics are generally characterized by limited therapeutic outcomes. The
serotonin 5‐HT1A receptor and the  adrenergic receptors are involved in central nociceptive
mechanisms with a pivotal role in the inhibitory descending pain pathway. Since their stimulation
may modulate the nervous signaling altered by neuropathies, the purpose of the present research is
the study of the combined activation of 5‐HT1A and  receptors by rationally designed imidazoline
ligands ((S)-1 and 2-5) in a rat model of neuropathic pain (chronic constriction injury - CCI). On
day 14 after nerve damage, the acute administration per os (p.o.) of low doses of (S)-(-)-1 (0.1-1
mg/kg) was able to significantly increase the pain threshold to mechanical noxious stimuli for more
th

Dedicated to Prof. Maria Pigini, who passed away on February 8 , 2016.
1

than 1h. (S)-(-)-1 efficacy was confirmed by the decrease of spontaneous pain evaluated as hind
limb weight bearing alterations. The clinically-used compound gabapentin (100 mg/kg p.o.) induced
a pain relieving effect similar to (S)-(-)-1 administered at 100 fold lower dose. In the same model,
the selected analogues, compounds 2-5 (1 mg/kg p.o.) were effective 30 min after administration. In
particular, 5 fully reverted the CCI-induced hypersensitivity. The pain relieving activity of 5 was
significantly prevented by the selective 5-HT1A receptor antagonist WAY 100635 (1 mg/kg
intraperitoneally, i.p.) and, at a lesser extent, by the 2 antagonist yohimbine (3 mg/kg i.p.). A novel
pharmacodynamic approach to the treatment of neuropathic pain is presented.
Keywords
Imidazoline, mixed 5HT1A/2agonists, CCI, neuropathy, chronic pain, gabapentin
Chemical compound studied in this article:
(S)-(-)-1 (PubChem CID 10333157); 2 (PubChem CID 11265631); 3 (PubChem CID 11211035); 4
(PubChem CID 44549156); 5 (PubChem CID 23622576).

1. Introduction
The treatment of neuropathic pain is heavily hampered by the pathological changes of the pain
pathways that characterized this condition (Garland, 2012). The use of opioids, fully active against
nociceptive pain, is unsatisfactory in neuropathic patients (McNicol et al., 2013; Sun et al., 2017),
in addition their long-term use is related to the development of pharmacodynamic tolerance
(Ballantyne and Shin, 2008; Dumas and Pollack, 2008). Non-analgesic drugs, like anti-depressants
or anti-epileptics, are currently used despite the partial efficacy and the significant side-effects
(Hurley et al., 2013).
The pathological aspects of neuropathic pain involve a maladaptive response of the nervous system
characterized by peripheral and central sensitization and spinal disinhibition. Disruption of the
2

balance of descending modulatory circuits to favour facilitation may promote and maintain chronic
pain (Von Hehn et al., 2012; Ossipov et al., 2010; Di Cesare Mannelli et al., 2016). This scenario is
consistent with the clinical success of drugs that enhance spinal monoaminergic activity, such as
serotonin/norepinephrine reuptake inhibitors (SNRIs), in the treatment of chronic pain states.
Among the serotonin receptors, the 5‐HT1A receptor has been one of the first to be
pharmacologically characterized (Fargin et al., 1988; Fargin et al., 19898). It is expressed in the
raphe nucleus and in several postsynaptic areas involved in central nociceptive mechanisms
(Kalipatnapu and Chattopadhyay, 2007). Full and partial 5-HT1A receptor agonists have shown to
be beneficial in pain treatments (Nadeson and Goodchild, 2002) arousing great interest as future
therapeutic agents (Muthuraman et al., 2014, Panczyk et al., 2015).
On the other side, among the receptors recognized by noradrenaline, 2 receptor activation decrease
the intracellular adenyl-cyclase activity through Gi or directly modify the activity of ion channels
(Summers and McMartin, 1993; Giovannoni et al., 2009). The three subtypes (2A, 2B and 2C) are
involved in different physiological functions and their pharmacological stimulation elicits anti-
hyperalgesic effects; the prototype agonist clonidine is currently in clinical use in spite of a not
optimal safety profile (Szczudlik et al., 2014). Besides clonidine, other 2-substituted imidazolines
are used in human or veterinary pain treatment (Giovannoni et al., 2009). Moreover, the multiplicity
of the effects produced by imidazoline compounds highlights the wide bioversatility of this scaffold
(Dardonville and Rozas, 2004; Krasavin, 2015).
It has been demonstrated that the (S)-(-) enantiomer of biphenyline (1, Fig.1), described as 2
receptor (Gentili et al., 2002) and 5-HT1A receptor (Del Bello et al., 2016) agonist, behaved as a
potent and long-lasting antinociceptive agent in algesiometric paradigms (Gentili et al., 2002).
Considering the important role played by both 5-HT1A and 2 receptors in nociception (Colpaert,
2006; Deseure et al., 2007; Di Cesare Mannelli et al., 2016), in the present research we examined
the pain relieving effect of (S)-(-)-1 in the rat model of peripheral mononeuropathy induced by
3

chronic constriction injury (CCI) of the sciatic nerve. Moreover, to obtain more information about
the role of 5-HT1A and 2 receptors in the neuropathic pain relieving effect of (S)-(-)-1, and
eventually improve its pharmacological efficacy, the structurally analogues 2-5 (Fig.1) have been
rationally selected to be tested in the same rat model of peripheral neuropathy.
2. Materials and Methods
2.1.Drugs
Compound (S)-(-)-1 ((S)-2-(1-([1,1'-biphenyl]-2-yloxy)ethyl)-4,5-dihydro-1H-imidazole) was
obtained treating methyl (S)-2-([1,1'-biphenyl]-2-yloxy)propanoate with ethylenediamine and
trimethyl aluminium at 40 °C in toluene (Gentili et al., 2002).
Compounds 2 (3-(2-(1-(4,5-dihydro-1H-imidazol-2-yl)ethoxy)phenyl)pyridine) and 3 (2-(1-((3'-
fluoro-[1,1'-biphenyl]-2-yl)oxy)ethyl)-4,5-dihydro-1H-imidazole) were obtained treating 2-(2-
(pyridin-3-yl)phenoxy)propanenitrile or 2-((3'-fluoro-[1,1'-biphenyl]-2-yl)oxy)propanenitrile,
respectively, with ethylenediamine in the presence of sodium methoxide (Gentili et al., 2004).
Compounds 4 (2-(1-(2-cyclopropylphenoxy)ethyl)-4,5-dihydro-1H-imidazole) and 5 (2-(1-(2-
cyclohexylphenoxy)ethyl)-4,5-dihydro-1H-imidazole) were obtained treating methyl 2-(2-
cyclopropylphenoxy)propanoate or methyl 2-(2-cyclohexylphenoxy)propanoate, respectively, with
ethylenediamine and trimethyl aluminium at 110 °C in toluene (Cardinaletti et al., 2009; Gentili et
al., 2008).
2.2. Animals
Male Sprague-Dawley rats (Envigo, Varese, Italy) weighing approximately 200–250 g were used.
Animals were housed in CeSAL (Centro Stabulazione Animali da Laboratorio, University of
Florence) and used at least 1 week after their arrival. Four rats were housed per cage (size 26 × 41
cm); animals were fed a standard laboratory diet and tap water ad libitum, and kept at 23 ± 1 °C
4

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 scientific 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 Animal Subjects Review Board of the University of Florence. Experiments involving
animals have been reported according to ARRIVE guidelines (McGrath and Lilley, 2015). All
efforts were made to minimize animal suffering and to reduce the number of animals used.
2.3. CCI-induced neuropathic pain model
Neuropathy was induced according to the procedure described by Bennett and Xie (1988). Briefly,
rats were anaesthetized with 2% isoflurane. Under aseptic conditions, the right (ipsilateral) common
sciatic nerve was exposed at the level of the middle thigh by blunt dissection. Proximal to the
trifurcation, the nerve was carefully freed from the surrounding connective tissue, and four chromic
cat gut ligatures (4-0, Ethicon, Norderstedt, Germany) were tied loosely around the nerve with
about a 1 mm spacing between ligatures. After hemostasis was confirmed, the incision was closed
in layers. The animals were allowed to recover from surgery and then housed one per cage with free
access to water and standard laboratory chow. Another group of rats was subjected to sham surgery
in which the sciatic nerve was only exposed but not ligated.
2.4. Pharmacological treatments
Compounds (S)-(-)-1 (0.1 and 1 mg/kg), 2-5 (1 mg/kg) and gabapentin (100 mg/kg) were suspended
in 1% carboxymethyl cellulose and administered per os (p.o.). WAY 100635 (1 mg/kg) and
yohimbine (3 mg/kg) were dissolved in saline solution and injected intraperitoneally (i.p.) 15 min
5

before the administration of compound 5. The dosages of (S)-(-)-1 were chosen on the basis of the
already evaluated antinociceptive ED50 after subcutaneous administration (Gentili et al., 2002) and
opportunely modified for the p.o. treatment. The doses of gabapentin, WAY 100635 and yohimbine
were chosen on the basis of previously published results (Di Cesare Mannelli et al., 2015; Wang et
al., 2013; Vergelli et al., 2015).
2.5. Paw-pressure test
The nociceptive threshold in the rat was determined with an analgesiometer (Ugo Basile, Varese,
Italy), according to the method described by Leighton et al. (1988). Briefly, constantly increasing
pressure was applied to a small area of the dorsal surface of the hind paw using a blunt conical
mechanical probe. Mechanical pressure was increased until vocalization or a withdrawal reflex
occurred while rats were lightly restrained. Vocalization or withdrawal reflex thresholds were
expressed in grams. Rats scoring below 40 g or over 75 g during the test before drug administration
were rejected (25%). For analgesia measures, mechanical pressure application was stopped at 120 g.
The pain threshold was evaluated before and 15, 30, 45, 60 and 90 min after compound
administration.
2.6. Incapacitance test
Weight-bearing changes were measured using an incapacitance apparatus (Linton Instrumentation,
Norfolk, UK) to detect changes in postural equilibrium after a hind limb injury (Bove et al., 2003).
Rats were trained to stand on their hind paws in a box with an inclined plane (65° from horizontal).
This box was placed above the incapacitance apparatus. This allowed us to independently measure
the weight that the animal applied on each hind limb. The value reported for each animal was the
mean of 5 consecutive measurements. In the absence of hind limb injury, rats applied an equal
weight on both hind limbs, indicating a postural equilibrium, whereas an unequal distribution of the
6

weight on hind limbs indicated a monolateral decreased pain threshold. Data are expressed as the
difference between the weight applied to the limb contralateral to the injury and the weight applied
to the ipsilateral one ( Weight).
2.7. Statistical analysis
Behavioral measurements were performed on 6 rats for each treatment carried out in 2 different
experimental sets. Results were expressed as means ± S.E.M. and the analysis of variance was
performed by Two-Way ANOVA. A Bonferroni’s significant difference procedure was used as
post-hoc comparison. All assessments were made by researchers blinded to cell or rat treatments.
Data were analyzed using the ‘‘Origin 8.1” software (OriginLab, Northampton, USA).
3. Results
The pain relieving effects of the compounds were evaluated in rats underwent CCI of the sciatic
nerve. This rat model of mononeuropathy provokes a painful syndrome beginning about 3 days
after nerve ligation and plateauing between 7 and 30 days (Pacini et al., 2010; Di Cesare Mannelli
et al., 2014). Fourteen days after injury, the response to a noxious mechanical stimulus was
measured by the Paw pressure test. In the CCI + vehicle group the weight tolerated on the ipsilateral
paw decreased to 45.3 ± 2.1 g in comparison to 67.3 ± 2.1 of control animals (sham + vehicle) (Fig.
2). (S)-(-)-1 (0.1 and 1 mg/kg p.o.) was able to reduce the hypersensitivity induced by the loose
ligation of the sciatic nerve. The anti-hyperalgesic activity took effect 15 min after administration.
(S)-(-)-1 (1 mg/kg, 30 min) showed an anti-hyperalgesic efficacy of 56%, considering as 100% the
complete reversion of pain till the threshold of control animals. The effect was still statistically
significant 60 min after injection and vanished at 90 min (Fig. 2). The response of the contralateral
paw was unaltered (data not shown) suggesting the lack of analgesic effects. Gabapentin was used
as a reference compound since its relevant clinical use in neuropathic patients (Szczudlik et al.,
7

2014). The common and accepted rat dosage of 100 mg/kg p.o. (Di Cesare Mannelli et al., 2015)
was active from 15 to 45 min after treatment inducing a relief slightly higher (74% of anti-
hyperalgesic efficacy) than (S)-(-)-1 administered at 100 fold lower dose (1 mg/kg) (Fig. 2).
As shown in Fig. 3, monolateral pain was also able to induce hind limb weight bearing alterations
(Incapacitance test). The difference between the weight burdened on the contralateral and the
ipsilateral paw increased in CCI + vehicle (55.3 ± 4.2 g) in comparison to sham + vehicle (7.3 ± 2.1
g). (S)-(-)-1 (1.0 mg/kg) significantly reduced the hind limb weight bearing alterations in
comparison to CCI + vehicle. The effect started 15 min after administration and vanished at 60 min.
A comparable effect was induced by the acute administration of 100 mg/kg gabapentin.
The imidazolines 2-5 were also effective in reducing the hypersensitivity induced by the loose
ligation of the sciatic nerve 30 min after a single administration p.o. of 1 mg/kg. The percentages of
anti-hyperalgesic efficacy (30 min) of 2, 3, and 4 are 37.5%, 52.5%, and 35%, respectively; whereas
5 reaches 95% (Fig. 4). The pain killer effect of 5 is long lasting in comparison to 2, 3 and 4 and the
efficacy is higher than (S)-(-)-1.
The co-administration of 5 with WAY 100635 (1 mg/kg i.p. 15 min before 5) greatly reduced the
pain relieving effect. In Fig. 5 (panel a), a full reversion of 5 activity is shown with the exception of
a breakthrough at 30 min. The blocking effect induced by yohimbine (3 mg/kg i.p. 15 min before 5)
is significant 15 and 30 min after treatment even if lesser in efficacy as well as duration (Fig. 5,
panel b). The combined administration of both the antagonists WAY 100635 (1 mg/kg i.p.) and
yohimbine (3 mg/kg i.p.), 15 min before 5, completely prevented the pain relieving effect of
compound 5 (Fig. 5, panel c).
4. Discussion
The descending pain modulatory system originates in the midbrain (periaqueductal gray) and
brainstem (rostroventromedial medulla) and projects to the spinal cord dorsal horn, powerfully
8

influencing the modulation of nociceptive information transmitted from the periphery to the brain
(Von Hehn et al., 2012). The role of noradrenaline in this system appears to be predominantly
inhibitory, while the role of serotonin appears to be bidirectional, mediating inhibitory as well as
excitatory effects (Ossipov et al., 2010). The increased activity of the descending noradrenergic
system and high extracellular levels of spinal noradrenaline has been linked to
antinociceptive/antiallodynic/antihyperalgesic effects in acute and neuropathic pain (Di Cesare
Mannelli et al., 2016). The 5‐HT1A receptor is expressed by all serotoninergic neurons (as
autoreceptors) and by many non serotoninergic neurons (as heteroreceptors) in pain-related areas of
the brain (Kalipatnapu and Chattopadhyay, 2007). As already mentioned, 5-HT1A receptor agonists
have shown to produce efficacious antinociceptive effect in rats (Nadeson and Goodchild, 2002). In
particular, the 5‐HT1A agonist F13640 provided the first evidence that the activation of this receptor
produces analgesia in a model of trigeminal neuropathic pain. It is characterized by long-term
analgesia in rodent models of peripheral neuropathic pain and anti-allodynic action in spinal cord
injured rat (Colpaert et al., 2002; Deseure et al., 2002). As regards the 2 adrenergic receptor, the
2A, 2B and 2C subtypes have been identified as potential contributors to nociceptive modulation
being located in key nodes of the nociceptive system. In the spinal cord, the 2 receptor stimulation
induces analgesia by post-synaptic hyperpolarization and presynaptic inhibition of the excitatory
transmission (Di Cesare Mannelli et al., 2016).
The double-face profile of the tested imidazoline compounds allows the simultaneous modulation of
neurotransmitter signals of great relevance in pain mechanism. In particular, the efficacy of the 5-
HT1A receptor and 2 receptor agonist (S)-(-)-1 in reducing the hypersensitivity and the hind limb
weight bearing alterations induced by the loose ligation of the sciatic nerve allows us to
hypothesize the involvement of both the receptor systems in its anti-neuropathic activity.
9

The experiments performed with the selected imidazolines 2-5 provided further information about
the role played by both 5-HT1A and 2 receptors. The compounds have been selected considering
their good but different affinities for 5-HT1A receptor and similar agonist activities at 2C subtype.
They all behave as agonists at 2A subtype, except for compound 4, which acts as an antagonist
(Table 1). Based on the observation that α2A subtype has been demonstrated to be critical for the
pain relieving actions of α2 receptor agonists in a mouse model of neuropathic pain (Malmberg et
al., 2001), this last compound might confirm such a finding from a pharmacological point of view.
Derivative 5, showing the highest affinity for 5-HT1A receptor and agonist activity at all the α2
receptor subtypes, is the most effective compound in reducing mechanical hypersensitivity.
Compared to 5, the lower efficacy of (S)-(-)-1, 2 and 3 in in vivo studies might be attributed to their
lower affinity for 5-HT1A receptor (Table 1). Though derivative 4 is characterized by 5-HT1A
receptor affinity similar to that of 5 (Table 1), it also proved to be less effective in reducing the
neuropathic pain. This behaviour might result from its α2A antagonist profile, supporting the finding
that α2A receptor subtype is essential for the anti-neuropathic effects mediated by α2 receptor
agonists (Malmberg et al., 2001). The comparable anti-neuropathic efficacy of the α2A antagonist 4
and the α2A agonist 2 might be due to the significantly higher affinity and agonist activity for 5-
HT1A receptor of 4 with respect to 2.The experiments performed with derivative 5 in the presence of
the 5-HT1A receptor antagonist WAY 100135 and the 2 receptor antagonist yohimbine confirm the
relevance of the positive modulation of both serotoninergic and adrenergic system in the
pharmacodynamic mechanism of such a compound. Therefore, the double 5-HT1A/α2 agonist profile
of the studied imidazoline derivatives can produce a fruitful modulation of the descending
antinociceptive pathway. Moreover, the observation that yohimbine reduces the pain relieving
effect at lesser extent than WAY 100635 suggests a predominance of the 5-HT1A component.
10

5. Conclusion
In conclusion, the present study (i) highlights that the bioversatile 2-imidazoline substituted
scaffold might be a valuable structure in the building of novel anti-neuropathic agents. (ii)
Noteworthy, the multifunctional agent (S)-(-)-1 is able to reduce neuropathic pain at the dose of 1.0
mg/kg, being 100 fold more potent than the clinically used gabapentin. (iii) Moreover, compounds
2-5 show different efficacies and potencies after per os treatment of CCI rats, providing useful
information on the role played by the 5-HT1A and 2 receptors in their neuropathic pain relieving
effect. In particular 5, showing the highest affinity for the 5-HT1A receptor and agonist activity at all
the α2 receptor subtypes, is the most effective compound in reducing mechanical hypersensitivity
through a mechanism significantly inhibited by the pharmacological blockade of 5-HT1A or 2
receptors. Therefore, the combined 5-HT1A/2 receptors activation might be considered a promising
pharmacodynamic approach to the treatment of neuropathic pain.
Acknowledgements and funding
This research was supported by the Italian Ministry of Instruction, University and Research
(MIUR); the University of Florence, and the University of Camerino (Fondo di Ateneo per la
Ricerca 2014-2015). The authors declare no conflict of interest.
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16

Figure captions
Fig. 1. Chemical structures of 1-5.
Fig. 2. Effect of acute administration of (S)-(-)-1 in comparison to gabapentin on CCI-induced
mechanical hypersensitivity. Peripheral neuropathy was induced by CCI of the right sciatic nerve
(ipsilateral); (S)-(-)-1 (0.1–1.0 mg/kg) was acutely administered p.o. on day 14 after surgery. The
Paw pressure test was used to measure the sensitivity to a mechanical stimulus 0, 15, 30, 45, 60 and
90 min after compound administration. Gabapentin (100 mg/kg, p.o.) was used as reference
compound; sham animals were treated with vehicle. Each value is the mean ± S.E.M. of 6 rats per
group, performed in 2 different experimental sets. **P<0.01 vs sham + vehicle; ^P<0.05 and
^^P<0.01 vs CCI + vehicle.
17

Fig. 3. Effect of acute administration of (S)-(-)-1 in comparison to gabapentin on CCI-induced
hyperalgesia. Peripheral neuropathy was induced by CCI of the right sciatic nerve (ipsilateral); (S)-
(-)-1 (0.1 - 1 mg/kg) was acutely administered p.o. on day 14 after surgery. Incapacitance test was
used to measure postural unbalance related to pain 0, 15, 30, 45, 60 and 90 min after compound
administration. Gabapentin (100 mg/kg, p.o.) was used as reference drug and sham animals were
treated with vehicle. Data are expressed as the difference between the weight applied to the limb
contralateral to the injury and the weight applied to the ipsilateral one ( Weight). Each value is the
mean ± S.E.M. of 6 rats per group, performed in 2 different experimental sets. **P<0.01 vs sham +
vehicle; ^P<0.05 and ^^P<0.01 vs CCI + vehicle.
Fig. 4. Effect of acute administration of compounds 2-5 in comparison to (S)-(-)-1 on CCI-induced
mechanical hypersensitivity. Peripheral neuropathy was induced by CCI of the right sciatic nerve
(ipsilateral), compounds were acutely administered p.o. (1.0 mg/kg) on day 14 after surgery. The
Paw pressure test was used to measure the sensitivity to a mechanical stimulus 0, 15, 30, 45, 60 and
90 min after compound administration. Sham animals were treated with vehicle. Each value is the
mean ± S.E.M. of 6 rats per group, performed in 2 different experimental sets. **P<0.01 vs sham +
18

vehicle; ^P<0.05 and ^^P<0.01 vs CCI + vehicle.
Fig. 5. Evaluation of the 5-HT1A receptor and 2-adrenoceptor involvement in the pain relieving
activity of compound 5. Peripheral neuropathy was induced by CCI of the right sciatic nerve
(ipsilateral). The Paw pressure test was performed on day 14 after surgery. 5 was acutely
administered p.o. (1.0 mg/kg), WAY 100635 (1 mg/kg) and yohimbine (3 mg/kg) were
administered i.p. 15 min before. Sham animals were treated with vehicle. Each value is the mean ±
S.E.M. of 6 rats per group, performed in 2 different experimental sets. Data of CCI + vehicle
groups are different (P<0.01) vs sham + vehicle; ^P<0.05 and ^^P<0.01 vs CCI + vehicle; °°P<0.01
vs CCI + 5 treated animals.
19

Table 1.a Affinity (pKi), Antagonist Potency (pKb), Agonist Potency (pEC50), Intrinsic Activity (ia)
on Human α2 receptor Subtypesb; Affinity (pKi), Agonist Potency (pD2), Relative Efficacy (%Emax
)
on Human 5-HT1A receptorc
α2A
α2B
α2C
5-HT1A
Compound
pEC50
(pKb)
pKi
ia
pKi
pEC50
ia
pKi
pEC50
ia
pKi
pD2
%Emax
1
7.32d
6.94d
0.70d 6.30d
0.80d 6.23d
6.19 d
0.50d 6.70d
0.65d 6.52d
7.24d
0.80d 7.34e 6.30e
0.90d 7.60e
120.2e
Biphenyline
(S)-(-)-1
7.04d
6.86f
7.83f
7.13d
6.50f
6.98f
6.16d
6.00f
NAf
7.73d
7.30f
7.22f
7.40g
7.68h
-
-
0.50f
0.58f
-
6.03
6.57f
6.51g
0.50f
-
7.19f
7.77f
1.00f
1.15f
7.10e 6.58e
7.60e 6.85e
79.34e
79.55e
92e
2
3
4
5
7.64g (7.00)g
7.13h 7.10h
5.50g
6.70h
0.70g 7.10g
0.70h 7.11h
0.90g 8.03e 7.24e
0.80h 8.25e
0.60h 6.72h
-
-
b
c
aThe data were expressed as means of 3-6 separate experiments. According to Del Bello et al., 2010. According to
Quaglia et al., 2008. dSee Crassous et al., 2007. eSee Del Bello et al., 2016. fSee Gentili et al., 2004. gSee Cardinaletti et
al., 2009. eSee Gentili et al., 2008.
20