Author’s Accepted Manuscript Antinociceptive properties of selective MT2 melatonin receptor partial agonists Martha López-Canul, Stefano Comai, Sergio Domínguez-López, Vinicio Granados-Soto, Gabriella Gobbi www.elsevier.com/locate/ejphar
PII: DOI: Reference:
S0014-2999(15)30139-4 http://dx.doi.org/10.1016/j.ejphar.2015.07.010 EJP70108
To appear in: European Journal of Pharmacology Received date: 13 March 2015 Revised date: 2 July 2015 Accepted date: 6 July 2015 Cite this article as: Martha López-Canul, Stefano Comai, Sergio DomínguezLópez, Vinicio Granados-Soto and Gabriella Gobbi, Antinociceptive properties of selective MT2 melatonin receptor partial agonists, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.07.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antinociceptive properties of selective MT2 melatonin receptor partial agonists
Martha López-Canul1,2*, Stefano Comai1*, Sergio Domínguez-López1, Vinicio GranadosSoto3, Gabriella Gobbi1*
1
Neurobiological Psychiatry Unit, Dept. Psychiatry, McGill University, Montreal, Canada
2
3
Universidad Veracruzana, Xalapa, Veracruz, México
Neurobiology of Pain Laboratory, Departamento de Farmacobiología, Cinvestav, Sede Sur, México, D.F., México * These authors have equally contributed
* Corresponding author Dr. Gabriella Gobbi: Neurobiological Psychiatry Unit Department, Psychiatry McGill University, 1033 Av des Pins Ouest, Room 220, Montreal, Qc H3A1A1 Canada. Tel: +1514-398-1290, Fax: +1-514-398-4866, email: [email protected]
Abstract (226 words) Melatonin is a neurohormone involved in the regulation of both acute and chronic pain whose mechanism is still not completely understood. We have recently demonstrated
1
that selective MT2 melatonin receptor partial agonists have antiallodynic properties in animal models of chronic neuropathic pain by modulating ON/OFF cells of the descending antinociceptive system. Here, we examined the antinociceptive properties of the selective MT2 melatonin receptor partial agonists N-{2-[(3methoxyphenyl)phenylamino]ethyl}acetamide (UCM765) and N-{2-[(3-bromophenyl)-(4fluorophenyl)amino]ethyl}acetamide (UCM924) in two animal models of acute and inflammatory pain: the hot-plate and formalin tests. UCM765 and UCM924 (5-40 mg/kg, s.c.) dose-dependently increased the temperature of the first hind paw lick in the hotplate test, and decreased the total time spent licking the injected hind paw in the formalin test. Antinociceptive effects of UCM765 and UCM924 were maximal at the dose of 20 mg/kg. At this dose, the effects of UCM765 and UCM924 were similar to those produced by 200 mg/kg acetaminophen in the hot-plate test, and by 3 mg/kg ketorolac or 150 mg/kg MLT in the formalin test. Notably, antinociceptive effects of the two MT2 partial agonists were blocked by the pre-treatment with the MT2 antagonist 4phenyl-2-propionamidotetralin (4P-PDOT, 10 mg/kg) in both paradigms. These results demonstrate the antinociceptive properties of UCM765 and UCM924 in acute and inflammatory pain models and corroborate the concept that MT2 melatonin receptor may be a novel target for analgesic drug development.
Keywords: Acute pain; Antinociception; Formalin test; Hot-plate test; Melatonin; MT2 receptor partial agonists.
1. Introduction (473 words)
2
Melatonin is a neurohormone produced in the pineal gland whose physiological effects occur mostly through the activation of two high-affinity G protein coupled receptors named MT1 and MT2 (Dubocovich et al., 2010). Melatonin regulates several physiological functions including sleep (Ochoa-Sanchez et al., 2014), mood (Comai and Gobbi, 2014), circadian rhythm (Cajochen et al., 2003) and pain (Ambriz-Tututi et al., 2009). In patients, it shows analgesic properties in fibromyalgia (Hussain et al., 2011), irritable bowel syndrome (Wilhelmsen et al., 2011) and migraine (Peres et al., 2004). Interestingly, melatonin levels change following acute pain (Nelson et al., 2001) and are also correlated to the intensities of responses to painful stimuli likely via a feedback mechanism between endogenous opioids and melatonin (Lissoni et al., 1986). The antinociceptive effects of melatonin have been also demonstrated in acute (Golombek et al., 1991; Lakin et al., 1981; Yu et al., 2000b), inflammatory (AmbrizTututi et al., 2009; Esposito et al., 2010; Pang et al., 2001) and neuropathic pain (Ambriz-Tututi and Granados-Soto, 2007; Arreola-Espino et al., 2007; Ulugol et al., 2006) models in mice and rats. The mechanisms of antinociceptive actions of melatonin are still unclear, although it has been hypothesized that the activation of GABAA, dopamine D2, opioid and MT2 melatonin receptors (Mantovani et al., 2006; Ambriz-Tututi et al., 2009; Comai and Gobbi, 2014) may be involved. Thus, oral administration of melatonin reduces formalin-induced nociception in diabetic rats, and these effects are partially prevented by the MT2 melatonin receptor antagonist K-185 or by the opioid receptor antagonist naltrexone (Arreola-Espino et al., 2007). In addition, oral or intrathecal melatonin administration in rats subjected to spinal nerve injury produces a MT2 and opioid receptors dependent antiallodynic effect (Ambriz-Tututi and Granados-
3
Soto, 2007). Consistent with these observations, MT2 melatonin receptors have been identified in cerebral structures related with pain such as thalamus, hypothalamus, trigeminal tract, trigeminal nucleus (Ambriz-Tututi et al., 2009; Lacoste et al., 2015), in glutamatergic neurons of the rostral ventrolateral periaqueductal gray (Lopez-Canul et al., 2015) and in the spinal cord (Pang et al., 1997). Recently, we have reported that the selective MT2 melatonin receptor partial agonist UCM924 has antiallodynic properties in animal models of neuropathic pain through modulation of brainstem descending antinociceptive pathways (Lopez-Canul et al., 2015). For the first time, here we tested whether the selective MT 2 receptor partial agonist and lead candidate N-{2-[(3-methoxyphenyl)phenylamino]ethyl}acetamide (UCM765) (MT1 receptors: pKi=8.38; MT2 receptors: pKi=10.18) (Rivara et al., 2007) and its analog N-{2-[(3-bromophenyl)-(4-fluorophenyl)amino]ethyl}acetamide (UCM924) (MT1 receptors: pKi=6.76; MT2 receptors: pKi=9.27) (Rivara et al., 2009) have antinociceptive properties in animal paradigms of acute thermal pain (Hunskaar et al., 1986) and persistent pain produced by formalin injection (Dubuisson and Dennis, 1977). The effects of UCM765 and UCM924 were also compared to those of melatonin (MT1 receptors: pKi=9.85; MT2 receptors: pKi=9.62) (Rivara et al., 2007) and ketorolac or acetaminophen. The participation of MT2 melatonin receptors in the antinociceptive effects of UCM765 and UCM924 were also determined.
4
2. Materials and methods 2.1. Experimental animals Male Wistar rats (200-250 g, Charles-River) were used for behavioral tests. All animals were housed at constant room temperature (20 ± 2°C) and humidity under a 12 h light/dark cycle (lights on at 7 AM) with food and water ad libitum. All experimental procedures were performed between 10 AM and 3 PM, were approved by the Animal Ethics Committee of McGill University (Canada), and were conducted according to the Ethical Guidelines for Investigation of Experimental Pain in Conscious Animals of the International Association for the Study of Pain.
2.2. Drugs and pharmacological treatments N-{2-[(3-methoxyphenyl)phenylamino]ethyl}acetamide (UCM765) and N-{2-[(3bromophenyl)-(4-fluorophenyl)amino]ethyl}acetamide (UCM924) (Rivara et al., 2007; Rivara et al., 2009) (5, 10, 20 and 40 mg/kg), melatonin (150 mg/kg), ketorolac (3 mg/kg), acetaminophen (200 mg/kg) (Sigma-Aldrich, Oakville, ON, Canada) and cis-4Phenyl-2-propionamidotetralin (4P-PDOT, 10 mg/kg) (Tocris, Ellisville, MO, SA) were dissolved in a vehicle composed of 70% dimethyl sulfoxide (MP Biochemicals, Solon, OH, USA) and 30% saline. The doses of UCM765, UCM924, and 4P-PDOT were chosen according to our recent study (Lopez-Canul et al., 2015). For the comparators melatonin, ketorolac and acetaminophen, we chose from the literature the doses that were able to induce antinociceptive effects in rats (Ambriz-Tututi and Granados-Soto, 2007; Jett et al., 1999; Sharp and Villano, 2012). Drugs were injected subcutaneously (s.c.; 0.5 ml) 30 min prior to behavioural tests. The selective MT2 melatonin receptor
5
antagonist 4P-PDOT (0.2 ml, s.c.) was injected 10 min prior the injection of vehicle, UCM765 or UCM924.
2.3. Pharmacological properties of UCM765 and UCM924 As previously described in detail (Rivara et al., 2007), the respective binding affinities of the melatonin receptors ligands UCM765 and UCM924 were measured using 2-[125I]iodomelatonin (100 pM) in competition experiments on cloned human MT 1 and MT2 melatonin receptors expressed in NIH3T3 rat fibroblasts. To define the functional activity of the two ligands at the MT1 and MT2 receptor subtypes, [35S]GTPS binding assays were also performed on the NIH3T3 cells expressing human-cloned MT1 or MT2 receptors (Rivara et al., 2007). Moreover, in vitro pharmacological assays covering a broad range of targets, including receptors, ion channels, transporters, enzymes and second messengers, were carried out by Cerep (France), according to experimental procedures described at www.cerep.fr.
2.4. Behavioral tests 2.4.1. Hot-plate test The hot-plate test was performed using an electronically controlled hot-plate (Ugo Basile, Italy) (Hunskaar et al., 1986). The initial temperature was 38°C and a near linear increase in temperature of 3°C per min was set. The temperature causing a fast hind paw lick was recorded as the nociceptive end-point. All tested animals had their first hind paw lick occurring at a temperature lower than 52°C which was set as the end
6
point (Hunskaar et al., 1986). After each session, the plate was cleaned with a wet cloth, and a fan was then used to cool the plate quickly.
2.4.2. Formalin-induced nociceptive test The formalin test was conducted following the method of Dubuisson and Dennis (1977). Rats were placed in the experimental chamber 60 min prior to the experiment for habituation. Animals were gently restrained while the dorsum of the hind paw was injected with 50 l of 1% formalin (37% formaldehyde solution further diluted in saline) into the dorsal surface of the right hind paw with a 30-gauge needle. Rats were immediately returned to the experimental chambers and nociceptive behaviour was observed for 60 min. Mirrors were placed behind the chamber to enable unhindered observation. Nociceptive behaviour was quantified as the cumulative time the animal spent licking the injected hind paw (Wheeler-Aceto and Cowan, 1993). As previously reported (Abbott et al., 1995), formalin-induced licking behaviour was biphasic: the initial acute phase (0–10 min) was followed by a relatively short quiescent period, which was then followed by a prolonged tonic response (15–60 min). At the end of the experiment, rats were killed with CO2.
2.5. Statistical Analysis SigmaPlot (version 12.0, Systat Software, San Jose, CA, USA) was used for data analysis. Results were expressed as mean ± S.E.M. of 5-6 animals per group. Statistical differences between treatments were calculated using two-way analysis of variance (ANOVA) for repeated measures or one-way ANOVA followed by the
7
Bonferroni post hoc test for multiple comparisons. The level of significance was set at P < 0.05.
3. Results 3.1. Binding affinity of the pro-lead UCM765 As reported in Table 1, the MT2 melatonin receptor partial agonist pro-lead UCM765 (1M) does not display significant affinity for a panel of receptors, ion channels and transporters, including those known for their involvement in pain, such as opioid, cannabinoid, serotonin and norepinephrine receptors, and K+, Na+ and Ca2+ channels. Pharmacokinetic studies indicate that a UCM924 dose of 20 mg/kg (s.c.) reaches a concentration between 2.5 and 3 nmol/g in the brain after 1 hour.
3.2. UCM765 and UCM924 showed antinociceptive properties in the hot-plate test UCM765 (Fig. 1A) and UCM924 (Fig. 1B) displayed antinociceptive effects in the hotplate test. A two-way repeated measures ANOVA employing the factors dose of UCM765 and time of testing showed a significant effect of UCM765 treatment (F3,20 = 23.57, P < 0.001), but not of time of testing (F2,40 = 0.48, P = 0.62), and no treatment x time interaction (F6,40 = 1.09, P = 0.38). Bonferroni post hoc test revealed that, compared to vehicle, UCM765 (20 and 40 mg/kg) produced an overall significant increase of the temperature at which the first hindpaw lick occurred (P
PII: DOI: Reference:
S0014-2999(15)30139-4 http://dx.doi.org/10.1016/j.ejphar.2015.07.010 EJP70108
To appear in: European Journal of Pharmacology Received date: 13 March 2015 Revised date: 2 July 2015 Accepted date: 6 July 2015 Cite this article as: Martha López-Canul, Stefano Comai, Sergio DomínguezLópez, Vinicio Granados-Soto and Gabriella Gobbi, Antinociceptive properties of selective MT2 melatonin receptor partial agonists, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.07.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antinociceptive properties of selective MT2 melatonin receptor partial agonists
Martha López-Canul1,2*, Stefano Comai1*, Sergio Domínguez-López1, Vinicio GranadosSoto3, Gabriella Gobbi1*
1
Neurobiological Psychiatry Unit, Dept. Psychiatry, McGill University, Montreal, Canada
2
3
Universidad Veracruzana, Xalapa, Veracruz, México
Neurobiology of Pain Laboratory, Departamento de Farmacobiología, Cinvestav, Sede Sur, México, D.F., México * These authors have equally contributed
* Corresponding author Dr. Gabriella Gobbi: Neurobiological Psychiatry Unit Department, Psychiatry McGill University, 1033 Av des Pins Ouest, Room 220, Montreal, Qc H3A1A1 Canada. Tel: +1514-398-1290, Fax: +1-514-398-4866, email: [email protected]
Abstract (226 words) Melatonin is a neurohormone involved in the regulation of both acute and chronic pain whose mechanism is still not completely understood. We have recently demonstrated
1
that selective MT2 melatonin receptor partial agonists have antiallodynic properties in animal models of chronic neuropathic pain by modulating ON/OFF cells of the descending antinociceptive system. Here, we examined the antinociceptive properties of the selective MT2 melatonin receptor partial agonists N-{2-[(3methoxyphenyl)phenylamino]ethyl}acetamide (UCM765) and N-{2-[(3-bromophenyl)-(4fluorophenyl)amino]ethyl}acetamide (UCM924) in two animal models of acute and inflammatory pain: the hot-plate and formalin tests. UCM765 and UCM924 (5-40 mg/kg, s.c.) dose-dependently increased the temperature of the first hind paw lick in the hotplate test, and decreased the total time spent licking the injected hind paw in the formalin test. Antinociceptive effects of UCM765 and UCM924 were maximal at the dose of 20 mg/kg. At this dose, the effects of UCM765 and UCM924 were similar to those produced by 200 mg/kg acetaminophen in the hot-plate test, and by 3 mg/kg ketorolac or 150 mg/kg MLT in the formalin test. Notably, antinociceptive effects of the two MT2 partial agonists were blocked by the pre-treatment with the MT2 antagonist 4phenyl-2-propionamidotetralin (4P-PDOT, 10 mg/kg) in both paradigms. These results demonstrate the antinociceptive properties of UCM765 and UCM924 in acute and inflammatory pain models and corroborate the concept that MT2 melatonin receptor may be a novel target for analgesic drug development.
Keywords: Acute pain; Antinociception; Formalin test; Hot-plate test; Melatonin; MT2 receptor partial agonists.
1. Introduction (473 words)
2
Melatonin is a neurohormone produced in the pineal gland whose physiological effects occur mostly through the activation of two high-affinity G protein coupled receptors named MT1 and MT2 (Dubocovich et al., 2010). Melatonin regulates several physiological functions including sleep (Ochoa-Sanchez et al., 2014), mood (Comai and Gobbi, 2014), circadian rhythm (Cajochen et al., 2003) and pain (Ambriz-Tututi et al., 2009). In patients, it shows analgesic properties in fibromyalgia (Hussain et al., 2011), irritable bowel syndrome (Wilhelmsen et al., 2011) and migraine (Peres et al., 2004). Interestingly, melatonin levels change following acute pain (Nelson et al., 2001) and are also correlated to the intensities of responses to painful stimuli likely via a feedback mechanism between endogenous opioids and melatonin (Lissoni et al., 1986). The antinociceptive effects of melatonin have been also demonstrated in acute (Golombek et al., 1991; Lakin et al., 1981; Yu et al., 2000b), inflammatory (AmbrizTututi et al., 2009; Esposito et al., 2010; Pang et al., 2001) and neuropathic pain (Ambriz-Tututi and Granados-Soto, 2007; Arreola-Espino et al., 2007; Ulugol et al., 2006) models in mice and rats. The mechanisms of antinociceptive actions of melatonin are still unclear, although it has been hypothesized that the activation of GABAA, dopamine D2, opioid and MT2 melatonin receptors (Mantovani et al., 2006; Ambriz-Tututi et al., 2009; Comai and Gobbi, 2014) may be involved. Thus, oral administration of melatonin reduces formalin-induced nociception in diabetic rats, and these effects are partially prevented by the MT2 melatonin receptor antagonist K-185 or by the opioid receptor antagonist naltrexone (Arreola-Espino et al., 2007). In addition, oral or intrathecal melatonin administration in rats subjected to spinal nerve injury produces a MT2 and opioid receptors dependent antiallodynic effect (Ambriz-Tututi and Granados-
3
Soto, 2007). Consistent with these observations, MT2 melatonin receptors have been identified in cerebral structures related with pain such as thalamus, hypothalamus, trigeminal tract, trigeminal nucleus (Ambriz-Tututi et al., 2009; Lacoste et al., 2015), in glutamatergic neurons of the rostral ventrolateral periaqueductal gray (Lopez-Canul et al., 2015) and in the spinal cord (Pang et al., 1997). Recently, we have reported that the selective MT2 melatonin receptor partial agonist UCM924 has antiallodynic properties in animal models of neuropathic pain through modulation of brainstem descending antinociceptive pathways (Lopez-Canul et al., 2015). For the first time, here we tested whether the selective MT 2 receptor partial agonist and lead candidate N-{2-[(3-methoxyphenyl)phenylamino]ethyl}acetamide (UCM765) (MT1 receptors: pKi=8.38; MT2 receptors: pKi=10.18) (Rivara et al., 2007) and its analog N-{2-[(3-bromophenyl)-(4-fluorophenyl)amino]ethyl}acetamide (UCM924) (MT1 receptors: pKi=6.76; MT2 receptors: pKi=9.27) (Rivara et al., 2009) have antinociceptive properties in animal paradigms of acute thermal pain (Hunskaar et al., 1986) and persistent pain produced by formalin injection (Dubuisson and Dennis, 1977). The effects of UCM765 and UCM924 were also compared to those of melatonin (MT1 receptors: pKi=9.85; MT2 receptors: pKi=9.62) (Rivara et al., 2007) and ketorolac or acetaminophen. The participation of MT2 melatonin receptors in the antinociceptive effects of UCM765 and UCM924 were also determined.
4
2. Materials and methods 2.1. Experimental animals Male Wistar rats (200-250 g, Charles-River) were used for behavioral tests. All animals were housed at constant room temperature (20 ± 2°C) and humidity under a 12 h light/dark cycle (lights on at 7 AM) with food and water ad libitum. All experimental procedures were performed between 10 AM and 3 PM, were approved by the Animal Ethics Committee of McGill University (Canada), and were conducted according to the Ethical Guidelines for Investigation of Experimental Pain in Conscious Animals of the International Association for the Study of Pain.
2.2. Drugs and pharmacological treatments N-{2-[(3-methoxyphenyl)phenylamino]ethyl}acetamide (UCM765) and N-{2-[(3bromophenyl)-(4-fluorophenyl)amino]ethyl}acetamide (UCM924) (Rivara et al., 2007; Rivara et al., 2009) (5, 10, 20 and 40 mg/kg), melatonin (150 mg/kg), ketorolac (3 mg/kg), acetaminophen (200 mg/kg) (Sigma-Aldrich, Oakville, ON, Canada) and cis-4Phenyl-2-propionamidotetralin (4P-PDOT, 10 mg/kg) (Tocris, Ellisville, MO, SA) were dissolved in a vehicle composed of 70% dimethyl sulfoxide (MP Biochemicals, Solon, OH, USA) and 30% saline. The doses of UCM765, UCM924, and 4P-PDOT were chosen according to our recent study (Lopez-Canul et al., 2015). For the comparators melatonin, ketorolac and acetaminophen, we chose from the literature the doses that were able to induce antinociceptive effects in rats (Ambriz-Tututi and Granados-Soto, 2007; Jett et al., 1999; Sharp and Villano, 2012). Drugs were injected subcutaneously (s.c.; 0.5 ml) 30 min prior to behavioural tests. The selective MT2 melatonin receptor
5
antagonist 4P-PDOT (0.2 ml, s.c.) was injected 10 min prior the injection of vehicle, UCM765 or UCM924.
2.3. Pharmacological properties of UCM765 and UCM924 As previously described in detail (Rivara et al., 2007), the respective binding affinities of the melatonin receptors ligands UCM765 and UCM924 were measured using 2-[125I]iodomelatonin (100 pM) in competition experiments on cloned human MT 1 and MT2 melatonin receptors expressed in NIH3T3 rat fibroblasts. To define the functional activity of the two ligands at the MT1 and MT2 receptor subtypes, [35S]GTPS binding assays were also performed on the NIH3T3 cells expressing human-cloned MT1 or MT2 receptors (Rivara et al., 2007). Moreover, in vitro pharmacological assays covering a broad range of targets, including receptors, ion channels, transporters, enzymes and second messengers, were carried out by Cerep (France), according to experimental procedures described at www.cerep.fr.
2.4. Behavioral tests 2.4.1. Hot-plate test The hot-plate test was performed using an electronically controlled hot-plate (Ugo Basile, Italy) (Hunskaar et al., 1986). The initial temperature was 38°C and a near linear increase in temperature of 3°C per min was set. The temperature causing a fast hind paw lick was recorded as the nociceptive end-point. All tested animals had their first hind paw lick occurring at a temperature lower than 52°C which was set as the end
6
point (Hunskaar et al., 1986). After each session, the plate was cleaned with a wet cloth, and a fan was then used to cool the plate quickly.
2.4.2. Formalin-induced nociceptive test The formalin test was conducted following the method of Dubuisson and Dennis (1977). Rats were placed in the experimental chamber 60 min prior to the experiment for habituation. Animals were gently restrained while the dorsum of the hind paw was injected with 50 l of 1% formalin (37% formaldehyde solution further diluted in saline) into the dorsal surface of the right hind paw with a 30-gauge needle. Rats were immediately returned to the experimental chambers and nociceptive behaviour was observed for 60 min. Mirrors were placed behind the chamber to enable unhindered observation. Nociceptive behaviour was quantified as the cumulative time the animal spent licking the injected hind paw (Wheeler-Aceto and Cowan, 1993). As previously reported (Abbott et al., 1995), formalin-induced licking behaviour was biphasic: the initial acute phase (0–10 min) was followed by a relatively short quiescent period, which was then followed by a prolonged tonic response (15–60 min). At the end of the experiment, rats were killed with CO2.
2.5. Statistical Analysis SigmaPlot (version 12.0, Systat Software, San Jose, CA, USA) was used for data analysis. Results were expressed as mean ± S.E.M. of 5-6 animals per group. Statistical differences between treatments were calculated using two-way analysis of variance (ANOVA) for repeated measures or one-way ANOVA followed by the
7
Bonferroni post hoc test for multiple comparisons. The level of significance was set at P < 0.05.
3. Results 3.1. Binding affinity of the pro-lead UCM765 As reported in Table 1, the MT2 melatonin receptor partial agonist pro-lead UCM765 (1M) does not display significant affinity for a panel of receptors, ion channels and transporters, including those known for their involvement in pain, such as opioid, cannabinoid, serotonin and norepinephrine receptors, and K+, Na+ and Ca2+ channels. Pharmacokinetic studies indicate that a UCM924 dose of 20 mg/kg (s.c.) reaches a concentration between 2.5 and 3 nmol/g in the brain after 1 hour.
3.2. UCM765 and UCM924 showed antinociceptive properties in the hot-plate test UCM765 (Fig. 1A) and UCM924 (Fig. 1B) displayed antinociceptive effects in the hotplate test. A two-way repeated measures ANOVA employing the factors dose of UCM765 and time of testing showed a significant effect of UCM765 treatment (F3,20 = 23.57, P < 0.001), but not of time of testing (F2,40 = 0.48, P = 0.62), and no treatment x time interaction (F6,40 = 1.09, P = 0.38). Bonferroni post hoc test revealed that, compared to vehicle, UCM765 (20 and 40 mg/kg) produced an overall significant increase of the temperature at which the first hindpaw lick occurred (P
