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Central and peripheral antinociceptive activity of 3-(2-oxopropyl)-3-hydroxy-2-oxindoles
Pharmacology, Biochemistry and Behavior 135 (2015) 13–19
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Central and peripheral antinociceptive activity of 3-(2-oxopropyl)-3-hydroxy-2-oxindoles Thais Biondino Sardella Giorno a,1, Yáskara L.L. Ballard a,1, Millena Santos Cordeiro a, Bárbara V. Silva b, Angelo C. Pinto b, Patricia Dias Fernandes a,⁎ a Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, Laboratório de Farmacologia da Dor e da Inflamação, Av. Carlos Chagas Filho, 373, Prédio do CCS, bloco J, sala 10, 21941-902, Rio de Janeiro, Brazil b Universidade Federal do Rio de Janeiro, Instituto de Química, Rio de Janeiro, Brazil
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Article history: Received 11 February 2015 Received in revised form 2 April 2015 Accepted 11 May 2015 Available online 16 May 2015 Keywords: Oxindoles Analgesia Antinociception Pain
a b s t r a c t Convolutamydine A has been shown to develop a significant antinociceptive effect. Here we demonstrated that new analogues (5-iodo-3-(2-oxopropyl)-3-hydroxy-2-oxindole (5-Iisa), 5-fluoro-3-(2-oxopropyl)-3-hydroxy2-oxindole (5-Fisa), 5-chloro-3-(2-oxopropyl)-3-hydroxy-2-oxindole (5-Clisa) and 5-methyl-3-(2-oxopropyl)3-hydroxy-2-oxindole (5-Meisa)), at 0.1–10 mg/kg doses, have significant peripheral and central antinociceptive effects in thermal and chemical models of nociception. Oral administered analogues demonstrated more pronounced antinociceptive effects than that obtained with the classical opioid drug morphine (5 mg/kg) in the first and second phases of formalin-induced licking. In the tail flick model, 5-Clisa and 5-Meisa antinociceptive effect was almost twice as that observed with the same dose of morphine. The concomitant administration of diverse antagonists and the analogues indicates that 5-Iisa effects involve the activation of opioid pathway. On the other hand, 5-Fisa and 5-Clisa have the participation of opioid, nitrergic, cholinergic adrenergic and serotoninergic pathways and 5-Meisa has the involvement of opioid, serotoninergic and cholinergic pathways. In conclusion, our results suggest that the new four analogues from Convolutamydine A have significant antinociceptive effects in thermal and chemical induced nociception and could be used in development of new drugs to be used in pain treatment with reduced side effects. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Pain is a hallmark of inflammation, and the therapeutic approaches to pain relief are based upon non-steroidal anti-inflammatory drugs or opiates. The improper use of either class of drugs gives rise to several unwanted effects such as ulcers, vomiting, tolerance, and dependence (Nakagawa and Kaneko, 2010; Zhao et al., 2011). The inevitable side effects associated with the use of these classes of drugs have led to the search for and synthesis of new efficient analgesics presenting little or no side effects in pain control. Convolutamydine A is a member of a family of oxindole alkaloids isolated from the Floridian marine bryozoan Amathia convoluta. This compound is interesting, as it has been described in the literature as having significant pharmacological activity. Several analogues have been synthesised from Convolutamydine A that demonstrate a diversity of effects (Kamano et al., 1995; Garden et al., 1997; Luppi et al., 2006; Cravotto et al., 2006). Our group described anti-inflammatory and/or
⁎ Corresponding author. Tel.: +55 21 39386442. E-mail address: [email protected] (P.D. Fernandes). 1 Contributed equally to this paper.
http://dx.doi.org/10.1016/j.pbb.2015.05.004 0091-3057/© 2015 Elsevier Inc. All rights reserved.
antinociceptive effects for a diversity of analogues (Matheus et al, 2007; Figueiredo et al, 2013; Fernandes et al., 2014). In this study we examine the antinociceptive effects of four synthesised 3-(2-oxopropyl)-3-hydroxy-2-oxindoles using thermal and inflammatory pain models in mice. We further evaluate the possible mechanisms of action of these substances with the hope of suggesting them as potential antinociceptive therapeutic agents. 2. Materials and methods 2.1. Animals All experiments were performed with male Swiss Webster mice (20–25 g) donated by Instituto Vital Brazil (Niterói, Brazil). The animals were maintained in a room with controlled temperature (22 ± 2 °C) on a 12-h light/dark cycle, with free access to food and water. All efforts were made to minimise animal suffering, to reduce the number of animals used. All animal experimental protocols are in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985), the UK Animals (Scientific Procedures) Act, 1986 and associated guidelines, the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH
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Publications No. 8023, revised 1978). The experimental protocols were also in accordance with the principles and guidelines adopted by the Brazilian College of Animal Experimentation (COBEA). Research protocols were approved by the Ethical Committee for Animal Research (Biomedical Science Institute/UFRJ) and received the approval number DFBCICB015-04/16. 2.2. General Acetylsalicylic acid (ASA), dexamethasone, atropine sulphate monohydrate, capsaicin, L-glutamic acid (glutamate), L-nitro arginine methyl ester (L-NAME), and carrageenan were purchased from Sigma (St. Louis, MO, USA), and formalin was purchased from Merck, Inc. Morphine sulphate and naloxone hydrochloride were kindly provided by Cristália (São Paulo, Brazil). All drugs were dissolved in phosphate buffer saline (PBS) just before use. Capsaicin was dissolved in 80% (v/v) ethanol plus 20% PBS. ASA (200 mg/kg, po) and dexamethasone (5 mg/kg, ip) were used as reference drugs. The negative control group was treated with the vehicle. 2.3. Synthesis of substances Isatins were suspended in acetone, and drops of diethylamine were added at room temperature, leading to 5-iodo-3-(2-oxopropyl)-3-hydroxy-2-oxindole (5-Iisa), 5-fluoro-3-(2-oxopropyl)-3-hydroxy-2oxindole (5-Fisa), 5-chloro-3-(2-oxopropyl)-3-hydroxy-2-oxindole (5-Clisa) and 5-methyl-3-(2-oxopropyl)-3-hydroxy-2-oxindole (5Meisa) (Fig. 1). The structures of the final compounds were determined by 1H-NMR and 13C-NMR, as described by Garden et al. (1997). 2.4. Administration of substances 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa were dissolved in dimethylsulphoxide (DMSO) to prepare stock solutions at 100 mg/ml. All substances were administered by oral gavage at doses of 0.1 to 10 mg/kg in a final volume of 0.1 ml of Tween 80 per animal. The control group was treated with vehicle (Tween 80/DMSO). In all experiments, the final concentration of DMSO or Tween 80 had no effect per se. 2.5. Acute toxicity Acute toxicity was determined according to the experimental model described by Lorke (1983). A single oral dose (150 mg/kg) of a substance was administered to a group of ten mice (five males and five females). Behavioural parameters, including convulsion, hyperactivity, sedation, grooming, loss of righting reflex, increased or decreased respiration, and food and water intake, were observed over a period of 5 days. After this period, the animals were sacrificed by cervical dislocation, their stomachs were removed, an incision was made along the greater curvature, and the number of ulcers (single or multiple erosions, ulcers or perforations) and degree of hyperaemia were counted.
Fig. 1. General structure of oxopropyl-oxindoles.
2.6. Formalin-induced licking model The procedure was similar to the method described by Hunskaar and Hole (1987) and with some modifications performed by Gomes et al. (2007). Animals received 20 μl of formalin (2.5% v/v) into the dorsal surface of the left hind paw. The time of licking the formalin-injected paw was immediately recorded during two phases: first phase (neurogenic pain response) between the moment of injection and 5 min and second phase (inflammatory pain response) between 15 and 30 min postinjection. The animals were pretreated with oral doses of each compound (0.1, 1 or 10 mg/kg), morphine (5 mg/kg), ASA (200 mg/kg), or vehicle (PBS) 60 min before the administration of formalin.
2.7. Glutamate- or capsaicin-induced nociception The procedure used for glutamate-induced nociception was described by Beirith et al. (2002) and adopted by Pinheiro et al. (2013), and for capsaicin-induced nociception the protocol described by Sakurada et al. (1998) was adopted by Pinheiro et al. (2013). Briefly, an intraplantar injection of glutamate (10 μl, 3.7 ng/paw) or capsaicin (20 μl; 1.6 μg/paw) was administered to the right hind paw. The animal was immediately placed in an individual box, and the time that the animal sustained licking or biting of the glutamate- or capsaicin-injected paw was recorded for 15 or 5 min, respectively. Mice were orally pretreated 60 min before the intraplantar injection of glutamate or capsaicin with each substance (0.1, 1 or 10 mg/kg) or vehicle. 2.8. Tail flick This test was performed according to Bem-Bassat et al. (1959) and adapted by Pinheiro et al. (2010). Briefly, mice were placed in a container tube, and one third of the tail was immersed in a water bath set to 50 ± 1 °C. The reaction time necessary for the animal to withdraw the tail was registered at several intervals of 20 min after the administration of a substance (0.1 to 10 mg/kg), morphine (5 mg/kg) or vehicle. Baseline was designated as the average of the reaction times obtained at 40 and 20 min before the administration of the substance, morphine or vehicle and defined as the normal reaction of the animal to thermal stimulus. If the animal did not withdraw their tail from the hot water for a period of time greater than three times the baseline (cut-off), their tail was manually removed to avoid any possible damage. Antinociception was quantified as an increase in baseline (IB) (%) calculated by the formula: IB = ((reaction time × 100) / baseline) − 100 or area under the curve (AUC) calculated by the formula based on the trapezoid rule of responses from 20 min after drug administration until the end of the experiment (Matthews et al., 1990): AUC = 20 × IB [(min 20) / 2 + (min 20 + min 40) / 2 + (min 40 + min 60) / 2… + (min 120) / 2]. The baselines values varied between 4 and 7 s.
2.9. Evaluation of the possible mechanism(s) of action of substances in the tail flick test To assess the possible participation of the opioid, cholinergic, noradrenergic, nitrergic, serotoninergic and cannabinoid systems in the antinociceptive effect of the substances, mice were pretreated with naloxone (opioid antagonist, 5 mg/kg, ip), mecamylamine (nicotinic antagonist, 2 mg/kg, ip), atropine (muscarinic antagonist, 1 mg/kg, ip), ondansetron (5-HT3 serotoninergic antagonist, 0.5 mg/kg, ip), yohimbine (alpha-2 adrenergic antagonist, 2 mg/kg, ip), or L-nitro arginine methyl ester (L-NAME, inhibitor of nitric oxide synthase, 3 mg/kg, ip) 15 min before treatment with a compound (10 mg/kg, po). Dose response curves for each antagonist were previously constructed and the dose that reduced the response to the agonist by 50% was chosen for these assays (Pinheiro et al., 2010; Figueiredo et al, 2013).
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2.10. Statistical analysis All experimental groups contained 6–10 mice. The results are presented as the mean ± SD. The significance of the difference between the mean values was evaluated with GraphPad Prism™ (version 5.00, GraphPad Software Inc., San Diego, CA, USA) by analysis of variance (ANOVA) followed by Bonferroni's tests. P values less than 0.05 were considered to indicate significant differences.
3. Results In the present study the antinociceptive efficacy of four 3-(2oxopropyl)-3-hydroxy-2-oxindoles was evaluated in several models of antinociception. Intraplantar injection of formalin produced a significant licking response in the formalin-injected paw of 46 ± 2.3 s (1st phase) and 145 ± 15 s (2nd phase). All three doses (0.1, 1 or 10 mg/kg) of each substance (5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa) significantly reduced both the 1st and 2nd phases of the formalin-licking response. It is also interesting to note that all substances had an effect in the 1st phase, and higher doses of 5-Iisa and 5-Fisa had a significant inhibitory effect (in 2nd phase) compared with the ASA-treated group (Fig. 2). To investigate the antinociceptive effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa we examined their effects on the tail flick model of thermal nociception. The results of Fig. 3 show that all compounds tested demonstrated an antinociceptive effect with differences occurring between each one. Effects higher than a 50% increase in baseline were observed with 1 and 10 mg/kg doses. For both doses the results varied between a 50% and 150% increase in baseline. The effects of 5-Iisa, 5-Fisa and 5-Clisa seem to reach a plateau at 80 or 100 min post-administration. When the time course curve was converted to an area under the curve (AUC) graph it was observed that 5-Clisa and 5-Meisa at 10 mg/kg had an effect significantly higher than that of morphine (Fig. 4).
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To further evaluate their antinociceptive activity the substances were tested in capsaicin- (Fig. 5A) and glutamate- (Fig. 5B) induced licking behaviour. Although all substances inhibited the licking response to both agonists, this inhibitory effect was more pronounced with 5-Clisa and 5-Meisa pretreatment. Moreover, the effects of both substances against the glutamate-induced response were stronger than those obtained with 5-Iisa or 5-Fisa. As 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa demonstrated significant peripheral and spinal antinociceptive effects, we further tried to evaluate the mechanism of action of the substances in the tail flick model. For this purpose we assessed the participation of endogenous systems (the opioid, cholinergic, adrenergic, nitrergic and serotoninergic systems) in the antinociceptive effects of the substances. Significant differences occurred between the inhibitory effects against each substance. Whereas naloxone (opioid receptor antagonist, 1 mg/kg, po) only reverted the antinociceptive effect of 5-Iisa, the effects of 5-Fisa and 5-Clisa were reverted by all antagonists used (L-NAME [inhibitor of the nitric oxide synthase enzymes, 3 mg/kg, po]; mecamylamine [nicotinic antagonist, 2 mg/kg, ip]; atropine [muscarinic antagonist, 1 mg/kg, ip]; ondansetron [5-HT3 serotoninergic antagonist, 0.5 mg/kg, ip]; yohimbine [alpha-2 adrenergic antagonist, 2 mg/kg, ip]). On the other hand, 5-Meisa's antinociceptive effect was inhibited by pretreatment of mice with naloxone, atropine or ondansetron (Fig. 6). We also evaluated the effects of antagonists in the model of formalin-induced licking response. Atropine did not demonstrate any significant effect versus either substance in either phase (1st or 2nd). Naloxone was the only antagonist that reverted the inhibitory effect of all the substances. Although L-NAME, mecamylamine and ondansetron reverted the 2nd phase of the licking response to all substances, the effects of these three antagonists in the 1st phase were variable (Fig. 7). In view of the significant results obtained in several models we also decided to further evaluate a possible toxic effect. Even when a dose of 150 mg/kg was used any kind of behavioural or histological alteration was observed (data not shown) suggesting that, at the doses used, these substances do not induce visual signs of toxicity. 4. Discussion
Fig. 2. Effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa on formalin-induced licking behaviour. Mice were pretreated with one of the substances, acetylsalicylic acid (ASA, 200 mg/kg), morphine (5 mg/kg) or vehicle. The results are presented as the mean ± SD (n = 6–10) of the time that the animal spent licking the formalin-injected paw. Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P b 0.05 compared to vehicletreated mice; #P b 0.05 compared to the morphine-treated group; P b 0.05 compared to the ASA-treated group.
In the present study we investigated the antinociceptive effects of four 3-(2-oxopropyl)-3-hydroxy-2-oxindoles using inflammatory and thermal pain models in mice. One interesting finding of this study was that when administered orally, all substances caused inhibition of the formalin-induced nociceptive response, even at doses as low as 0.1 mg/kg. Moreover, these effects were observed in both the 1st and 2nd phases. After subcutaneous injection of formalin an array of stereotyped responses occur that characterise the model as a useful methodology for studying acute and tonic pain. The first phase (or neurogenic) is due to stimulation of nociceptors, whereas the second phase (or inflammatory) is mediated by peripheral and spinal cord sensitisation (Hunskaar and Hole, 1987; Tjølsen et al., 1992). It was also shown that several mediators, such as amino acids, prostaglandin E2, nitric oxide (NO), substance P (SP), serotonin (5-HT) and histamine are increased at the spinal level (Tjølsen et al., 1992; Parada et al., 2001). Our results indicate that the four substances may act through nociceptors (because inhibition of the first phase was observed) or through altered sensitisation of the peripheral or spinal cord. It is also possible that a reduction in some of the inflammatory mediators liberated in the paw occurs. The administration of L-NAME (a NO synthase inhibitor) significantly reversed the antinociceptive effect induced by the oxindoles in the formalininduced licking behaviour. These data support the idea that the substances produce their antinociceptive effects through the involvement of nitric oxide pathways, as is the case for some anti-inflammatory drugs (Aguirre-Bañuelos and Lozano-Cuenca et al., 2005). Ondansetron (a 5-HT3 receptor antagonist) also reversed the effects of all oxindoles suggesting the involvement of the 5-HT3 receptor subtype pathway in
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Fig. 3. Effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa in the tail flick model in mice. Mice were pretreated with one of the substances, morphine (5 mg/kg) or vehicle. The results are presented as the mean ± SD (n = 6–10) of the increase in baseline. Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P b 0.05 when comparing treated mice to vehicletreated mice. Where no error bars are shown it is because they are smaller than the symbol.
the antinociceptive action of the substances. This finding corroborates other data showing that 5-HT receptor antagonists reduce the second phase of formalin-induced aversive responses without affecting the first one (Oyama et al., 2002; Bardin et al., 2001). Putative inflammatory mediators such as glutamate and capsaicin are also able to induce nociceptive pain responses after injection into the paw (Carstens, 1997; Hong and Abott, 1994). Based on this evidence, we sought to determine whether the four compounds inhibit the nociceptive response caused by intraplantar injection of glutamate or capsaicin into the mouse hindpaw. Our data demonstrate that the compounds produced a significant inhibition of the licking response
Fig. 4. Effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa in the tail flick model in mice. Mice were pretreated with one of the substances, morphine (5 mg/kg) or vehicle. The results are presented as the mean ± SD (n = 6–10) of the area under the curve (AUC). Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P b 0.05 when comparing treated mice to vehicle-treated mice and #P b 0.05 when comparing substance-treated mice with morphine-treated animals.
Fig. 5. Effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa on capsaicin (A) and glutamate (B) induced licking behaviour. Mice were orally pretreated with each substance or vehicle 60 min before capsaicin or glutamate injection into their paw. The results are presented as the mean ± SD (n = 6–10) of the time that the animal spent licking the capsaicin- or glutamate-injected paw. Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P b 0.05 when comparing treated mice to vehicle-treated animals.
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Fig. 6. Effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa in the tail flick model. Mice were pretreated with naloxone (opioid antagonist, 5 mg/kg, ip), mecamylamine (nicotinic antagonist, 2 mg/kg, ip), atropine (muscarinic antagonist, 1 mg/kg, ip), ondansetron (5-HT3 serotoninergic antagonist, 0.5 mg/kg, ip), yohimbine (alpha-2 adrenergic antagonist, 2 mg/kg, ip) or L-nitro arginine methyl ester (L-NAME, inhibitor of nitric oxide synthase, 3 mg/kg, ip) 15 min before oral administration of substances (10 mg/kg, po) or vehicle. The results are presented as the mean ± SD (n = 6–10) of the area under the curve (AUC). Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P b 0.05 when comparing treated mice to vehicle-treated mice and #P b 0.05 when comparing antagonist-treated groups with substances-treated animals.
caused by intraplantar injection of both mediators into the hindpaw. This inhibitory effect seems to be more pronounced in animals whose paws were injected with glutamate. Thus, the present results indicate that, at least in part, the antinociceptive action caused by oxindoles
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could be due to an interaction with the glutamatergic system. Capsaicin acts through vanilloid receptor type-1 (TRPV-1) expressed on nociceptive fibres and in the dorsal root ganglion of the spinal cord, trigeminal ganglia and central nervous system (Jancso, 1978; Oyama et al., 2002). The activation of TRPV1 receptors is also mediated by the release of neurotransmitters (i.e., glutamate and substance P), an effect that can participate in nociceptive processing (Afrah et al., 2001; Medvedeva et al., 2008). Glutamate is another neurotransmitter involved in nociceptive transmission, the development and maintenance of a nociceptive response through activation of ionotropic or metabotropic receptors, resulting in nociceptor sensitisation (Aanonsen and Wilcox, 1989; Budai et al., 1995; Carlton, 2001). An intraplantar injection of glutamate also induces the release of excitatory amino acids, PGE2, NO and kinins (Beirith et al., 2002; Sakurada et al., 1998) and promotes the activation of sensitive fibres that induce the release of several substances in the dorsal horn, which also can activate the TRPV1 receptor in the spinal cord (Julius and Basbaum, 2001; Millan, 2002). Our results are in accordance with previous data because the oxindoles reduce the nociceptive effects evoked by both glutamate and capsaicin. These results indicate that the antinociceptive effect of our substances involves, at least in part, the glutamatergic system. In addition to the results obtained in the capsaicin-induced nociception test, it is possible that they could produce antinociception by regulating TRPV1 receptors, thus contributing to the modulation of nociceptive transmission. To assess whether there would be spinal involvement in the spinal effect of oxindoles, we evaluated their antinociceptive effects in the tail flick model. All four substances had effects comparable to morphine with slight differences. The opioid reached its maximal effect at 80 min post-administration and decayed thereafter, whereas oxindoles reached their maximal effect sooner (i.e., 20 or 30 min) and maintained a plateau. It is well known that crosstalk between the adrenergic, opioidergic, nitrergic and serotoninergic pathways occurs at central and peripheral levels. For example, opioid and adrenergic receptors are located at the locus coeruleus and alpha-2 adrenergic receptors are localised on capsaicin-sensitive afferent fibres (Chen et al., 2007; Li and Eisenach, 2001; Fairbanks et al., 2002; Stein et al., 2009). This fact could explain the observation that all the antagonists used were able to reverse the antinociceptive effects of 5-Fisa and 5-Clisa in the tail flick model. It could be that the presence of fluoro or chloro atoms in their structures confers better solubility, facilitating their access to central sites of action. Behavioural alterations such as motor uncoordination and sedation might be misinterpreted as analgesia and produce false positive effects (Tabarelli et al., 2004). In the present study, oral administration of oxindoles did not cause motor impairment as evaluated by either forced locomotion in the rotarod or spontaneous locomotion in the open-field test. Thus, the possibility that the antinociceptive effect of the compounds tested is due to any degree of motor impairment or sedation is very low. We also observed that DMSO per se had any toxic or antiinflammatory/antinociceptive effect. Colucci et al. (2008) had demonstrated that the solvent has an antinociceptive effect when administered by different routes. However in that paper authors used a volume of 0.1 ml DMSO to each animal. In the present study we used a volume of 0.3 μl DMSO per animal (corresponding to the higher dose used, i.e., 10 mg/kg), a volume 333-fold lower than that used in the paper, demonstrating that this volume has any effect. We had previously showed that other analogue of Convolutamydine A, with a bromide atom at position 5, had significant effect in inflammatory (Fernandes et al., 2014) or antinociceptive models (Figueiredo et al., 2013). In those papers the effects were observed with doses ranging from 0.01 to 30 mg/kg. To the formalin-induced licking behaviour the molecule with a 5-Br substituent did not demonstrate any effect at 1st phase, and a significant effect at 2nd phase of the model was observed with 1 or 10 mg/kg doses. In the present work the new substances differ in the substituents. Either iodine, fluoride, chloride or methyl substituents at position 5 increase the activity of the molecule.
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Fig. 7. Effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa in the formalin-induced licking model. Mice were pretreated with atropine (muscarinic antagonist, 1 mg/kg, ip), naloxone (opioid antagonist, 5 mg/kg, ip), mecamylamine (nicotinic antagonist, 2 mg/kg, ip), yohimbine (alpha-2 adrenergic antagonist, 2 mg/kg, ip), L-nitro arginine methyl ester (L-NAME, inhibitor of nitric oxide synthase, 3 mg/kg, ip) or ondansetron (5-HT3 serotoninergic antagonist, 0.5 mg/kg, ip), 15 min before oral administration of substances (10 mg/kg, po) or vehicle. The results are presented as the mean ± SD (n = 6–10) of the time that mice spent licking the formalin-injected paw. Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P b 0.05 when comparing treated mice to vehicle-treated mice and #P b 0.05 when comparing antagonist-treated groups with substances-treated animals.
In this regard these substances demonstrate a significant effect even in the 1st phase of formalin-induced licking. All of them also demonstrate an effect in the hot plate model at doses of 0.1 mg/kg. It may be that these substituents confer stability to the molecule or facilitate the interaction between the molecule and the biological target increasing its effects. In conclusion, the data from the present study show that oxindoles exert pronounced systemic antinociception in chemical (formalin, glutamate and capsaicin-induced pain) and spinal (tail flick) models of nociception. In addition, the antinociceptive effects of the oxindoles were antagonised by L-NAME, naloxone, and ondansetron, but not by atropine in the formalin test (Table 1). Thus, these results suggest that oxindoles are able to produce antinociception by the activation of the nitric oxide, opioid or serotoninergic pathways.
Table 1 A summary of the possible mechanism of action to 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa. Receptor/System involved Opioid
NO
Muscarinic
Nicotinic
α2-adrenergic
5HT3
Substance
F
TF
F
TF
F
TF
F
TF
F
TF
F
TF
5-Iisa 5-Fisa 5-Clisa 5-Meisa
Y Y Y Y
Y Y Y Y
Y Y Y Y
– Y Y –
– – – –
– Y Y Y
– Y Y Y
– Y Y –
– – – –
– Y Y –
Y Y Y Y
– Y Y Y
F, formalin-induced licking; TF, tail flick; –, without effect.
Acknowledgments We would like to thank Mr. Alan Minho for technical assistance and Instituto Vital Brazil (Niterói, Brazil) for donation of mice. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant support and fellowship to PDF), Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ, grant support to PDF), and Conselho de Administração de Pessoal de Ensino Superior (CAPES, fellowship to TBSG and YLLB).
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Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Central and peripheral antinociceptive activity of 3-(2-oxopropyl)-3-hydroxy-2-oxindoles Thais Biondino Sardella Giorno a,1, Yáskara L.L. Ballard a,1, Millena Santos Cordeiro a, Bárbara V. Silva b, Angelo C. Pinto b, Patricia Dias Fernandes a,⁎ a Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, Laboratório de Farmacologia da Dor e da Inflamação, Av. Carlos Chagas Filho, 373, Prédio do CCS, bloco J, sala 10, 21941-902, Rio de Janeiro, Brazil b Universidade Federal do Rio de Janeiro, Instituto de Química, Rio de Janeiro, Brazil
a r t i c l e
i n f o
Article history: Received 11 February 2015 Received in revised form 2 April 2015 Accepted 11 May 2015 Available online 16 May 2015 Keywords: Oxindoles Analgesia Antinociception Pain
a b s t r a c t Convolutamydine A has been shown to develop a significant antinociceptive effect. Here we demonstrated that new analogues (5-iodo-3-(2-oxopropyl)-3-hydroxy-2-oxindole (5-Iisa), 5-fluoro-3-(2-oxopropyl)-3-hydroxy2-oxindole (5-Fisa), 5-chloro-3-(2-oxopropyl)-3-hydroxy-2-oxindole (5-Clisa) and 5-methyl-3-(2-oxopropyl)3-hydroxy-2-oxindole (5-Meisa)), at 0.1–10 mg/kg doses, have significant peripheral and central antinociceptive effects in thermal and chemical models of nociception. Oral administered analogues demonstrated more pronounced antinociceptive effects than that obtained with the classical opioid drug morphine (5 mg/kg) in the first and second phases of formalin-induced licking. In the tail flick model, 5-Clisa and 5-Meisa antinociceptive effect was almost twice as that observed with the same dose of morphine. The concomitant administration of diverse antagonists and the analogues indicates that 5-Iisa effects involve the activation of opioid pathway. On the other hand, 5-Fisa and 5-Clisa have the participation of opioid, nitrergic, cholinergic adrenergic and serotoninergic pathways and 5-Meisa has the involvement of opioid, serotoninergic and cholinergic pathways. In conclusion, our results suggest that the new four analogues from Convolutamydine A have significant antinociceptive effects in thermal and chemical induced nociception and could be used in development of new drugs to be used in pain treatment with reduced side effects. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Pain is a hallmark of inflammation, and the therapeutic approaches to pain relief are based upon non-steroidal anti-inflammatory drugs or opiates. The improper use of either class of drugs gives rise to several unwanted effects such as ulcers, vomiting, tolerance, and dependence (Nakagawa and Kaneko, 2010; Zhao et al., 2011). The inevitable side effects associated with the use of these classes of drugs have led to the search for and synthesis of new efficient analgesics presenting little or no side effects in pain control. Convolutamydine A is a member of a family of oxindole alkaloids isolated from the Floridian marine bryozoan Amathia convoluta. This compound is interesting, as it has been described in the literature as having significant pharmacological activity. Several analogues have been synthesised from Convolutamydine A that demonstrate a diversity of effects (Kamano et al., 1995; Garden et al., 1997; Luppi et al., 2006; Cravotto et al., 2006). Our group described anti-inflammatory and/or
⁎ Corresponding author. Tel.: +55 21 39386442. E-mail address: [email protected] (P.D. Fernandes). 1 Contributed equally to this paper.
http://dx.doi.org/10.1016/j.pbb.2015.05.004 0091-3057/© 2015 Elsevier Inc. All rights reserved.
antinociceptive effects for a diversity of analogues (Matheus et al, 2007; Figueiredo et al, 2013; Fernandes et al., 2014). In this study we examine the antinociceptive effects of four synthesised 3-(2-oxopropyl)-3-hydroxy-2-oxindoles using thermal and inflammatory pain models in mice. We further evaluate the possible mechanisms of action of these substances with the hope of suggesting them as potential antinociceptive therapeutic agents. 2. Materials and methods 2.1. Animals All experiments were performed with male Swiss Webster mice (20–25 g) donated by Instituto Vital Brazil (Niterói, Brazil). The animals were maintained in a room with controlled temperature (22 ± 2 °C) on a 12-h light/dark cycle, with free access to food and water. All efforts were made to minimise animal suffering, to reduce the number of animals used. All animal experimental protocols are in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985), the UK Animals (Scientific Procedures) Act, 1986 and associated guidelines, the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH
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Publications No. 8023, revised 1978). The experimental protocols were also in accordance with the principles and guidelines adopted by the Brazilian College of Animal Experimentation (COBEA). Research protocols were approved by the Ethical Committee for Animal Research (Biomedical Science Institute/UFRJ) and received the approval number DFBCICB015-04/16. 2.2. General Acetylsalicylic acid (ASA), dexamethasone, atropine sulphate monohydrate, capsaicin, L-glutamic acid (glutamate), L-nitro arginine methyl ester (L-NAME), and carrageenan were purchased from Sigma (St. Louis, MO, USA), and formalin was purchased from Merck, Inc. Morphine sulphate and naloxone hydrochloride were kindly provided by Cristália (São Paulo, Brazil). All drugs were dissolved in phosphate buffer saline (PBS) just before use. Capsaicin was dissolved in 80% (v/v) ethanol plus 20% PBS. ASA (200 mg/kg, po) and dexamethasone (5 mg/kg, ip) were used as reference drugs. The negative control group was treated with the vehicle. 2.3. Synthesis of substances Isatins were suspended in acetone, and drops of diethylamine were added at room temperature, leading to 5-iodo-3-(2-oxopropyl)-3-hydroxy-2-oxindole (5-Iisa), 5-fluoro-3-(2-oxopropyl)-3-hydroxy-2oxindole (5-Fisa), 5-chloro-3-(2-oxopropyl)-3-hydroxy-2-oxindole (5-Clisa) and 5-methyl-3-(2-oxopropyl)-3-hydroxy-2-oxindole (5Meisa) (Fig. 1). The structures of the final compounds were determined by 1H-NMR and 13C-NMR, as described by Garden et al. (1997). 2.4. Administration of substances 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa were dissolved in dimethylsulphoxide (DMSO) to prepare stock solutions at 100 mg/ml. All substances were administered by oral gavage at doses of 0.1 to 10 mg/kg in a final volume of 0.1 ml of Tween 80 per animal. The control group was treated with vehicle (Tween 80/DMSO). In all experiments, the final concentration of DMSO or Tween 80 had no effect per se. 2.5. Acute toxicity Acute toxicity was determined according to the experimental model described by Lorke (1983). A single oral dose (150 mg/kg) of a substance was administered to a group of ten mice (five males and five females). Behavioural parameters, including convulsion, hyperactivity, sedation, grooming, loss of righting reflex, increased or decreased respiration, and food and water intake, were observed over a period of 5 days. After this period, the animals were sacrificed by cervical dislocation, their stomachs were removed, an incision was made along the greater curvature, and the number of ulcers (single or multiple erosions, ulcers or perforations) and degree of hyperaemia were counted.
Fig. 1. General structure of oxopropyl-oxindoles.
2.6. Formalin-induced licking model The procedure was similar to the method described by Hunskaar and Hole (1987) and with some modifications performed by Gomes et al. (2007). Animals received 20 μl of formalin (2.5% v/v) into the dorsal surface of the left hind paw. The time of licking the formalin-injected paw was immediately recorded during two phases: first phase (neurogenic pain response) between the moment of injection and 5 min and second phase (inflammatory pain response) between 15 and 30 min postinjection. The animals were pretreated with oral doses of each compound (0.1, 1 or 10 mg/kg), morphine (5 mg/kg), ASA (200 mg/kg), or vehicle (PBS) 60 min before the administration of formalin.
2.7. Glutamate- or capsaicin-induced nociception The procedure used for glutamate-induced nociception was described by Beirith et al. (2002) and adopted by Pinheiro et al. (2013), and for capsaicin-induced nociception the protocol described by Sakurada et al. (1998) was adopted by Pinheiro et al. (2013). Briefly, an intraplantar injection of glutamate (10 μl, 3.7 ng/paw) or capsaicin (20 μl; 1.6 μg/paw) was administered to the right hind paw. The animal was immediately placed in an individual box, and the time that the animal sustained licking or biting of the glutamate- or capsaicin-injected paw was recorded for 15 or 5 min, respectively. Mice were orally pretreated 60 min before the intraplantar injection of glutamate or capsaicin with each substance (0.1, 1 or 10 mg/kg) or vehicle. 2.8. Tail flick This test was performed according to Bem-Bassat et al. (1959) and adapted by Pinheiro et al. (2010). Briefly, mice were placed in a container tube, and one third of the tail was immersed in a water bath set to 50 ± 1 °C. The reaction time necessary for the animal to withdraw the tail was registered at several intervals of 20 min after the administration of a substance (0.1 to 10 mg/kg), morphine (5 mg/kg) or vehicle. Baseline was designated as the average of the reaction times obtained at 40 and 20 min before the administration of the substance, morphine or vehicle and defined as the normal reaction of the animal to thermal stimulus. If the animal did not withdraw their tail from the hot water for a period of time greater than three times the baseline (cut-off), their tail was manually removed to avoid any possible damage. Antinociception was quantified as an increase in baseline (IB) (%) calculated by the formula: IB = ((reaction time × 100) / baseline) − 100 or area under the curve (AUC) calculated by the formula based on the trapezoid rule of responses from 20 min after drug administration until the end of the experiment (Matthews et al., 1990): AUC = 20 × IB [(min 20) / 2 + (min 20 + min 40) / 2 + (min 40 + min 60) / 2… + (min 120) / 2]. The baselines values varied between 4 and 7 s.
2.9. Evaluation of the possible mechanism(s) of action of substances in the tail flick test To assess the possible participation of the opioid, cholinergic, noradrenergic, nitrergic, serotoninergic and cannabinoid systems in the antinociceptive effect of the substances, mice were pretreated with naloxone (opioid antagonist, 5 mg/kg, ip), mecamylamine (nicotinic antagonist, 2 mg/kg, ip), atropine (muscarinic antagonist, 1 mg/kg, ip), ondansetron (5-HT3 serotoninergic antagonist, 0.5 mg/kg, ip), yohimbine (alpha-2 adrenergic antagonist, 2 mg/kg, ip), or L-nitro arginine methyl ester (L-NAME, inhibitor of nitric oxide synthase, 3 mg/kg, ip) 15 min before treatment with a compound (10 mg/kg, po). Dose response curves for each antagonist were previously constructed and the dose that reduced the response to the agonist by 50% was chosen for these assays (Pinheiro et al., 2010; Figueiredo et al, 2013).
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2.10. Statistical analysis All experimental groups contained 6–10 mice. The results are presented as the mean ± SD. The significance of the difference between the mean values was evaluated with GraphPad Prism™ (version 5.00, GraphPad Software Inc., San Diego, CA, USA) by analysis of variance (ANOVA) followed by Bonferroni's tests. P values less than 0.05 were considered to indicate significant differences.
3. Results In the present study the antinociceptive efficacy of four 3-(2oxopropyl)-3-hydroxy-2-oxindoles was evaluated in several models of antinociception. Intraplantar injection of formalin produced a significant licking response in the formalin-injected paw of 46 ± 2.3 s (1st phase) and 145 ± 15 s (2nd phase). All three doses (0.1, 1 or 10 mg/kg) of each substance (5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa) significantly reduced both the 1st and 2nd phases of the formalin-licking response. It is also interesting to note that all substances had an effect in the 1st phase, and higher doses of 5-Iisa and 5-Fisa had a significant inhibitory effect (in 2nd phase) compared with the ASA-treated group (Fig. 2). To investigate the antinociceptive effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa we examined their effects on the tail flick model of thermal nociception. The results of Fig. 3 show that all compounds tested demonstrated an antinociceptive effect with differences occurring between each one. Effects higher than a 50% increase in baseline were observed with 1 and 10 mg/kg doses. For both doses the results varied between a 50% and 150% increase in baseline. The effects of 5-Iisa, 5-Fisa and 5-Clisa seem to reach a plateau at 80 or 100 min post-administration. When the time course curve was converted to an area under the curve (AUC) graph it was observed that 5-Clisa and 5-Meisa at 10 mg/kg had an effect significantly higher than that of morphine (Fig. 4).
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To further evaluate their antinociceptive activity the substances were tested in capsaicin- (Fig. 5A) and glutamate- (Fig. 5B) induced licking behaviour. Although all substances inhibited the licking response to both agonists, this inhibitory effect was more pronounced with 5-Clisa and 5-Meisa pretreatment. Moreover, the effects of both substances against the glutamate-induced response were stronger than those obtained with 5-Iisa or 5-Fisa. As 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa demonstrated significant peripheral and spinal antinociceptive effects, we further tried to evaluate the mechanism of action of the substances in the tail flick model. For this purpose we assessed the participation of endogenous systems (the opioid, cholinergic, adrenergic, nitrergic and serotoninergic systems) in the antinociceptive effects of the substances. Significant differences occurred between the inhibitory effects against each substance. Whereas naloxone (opioid receptor antagonist, 1 mg/kg, po) only reverted the antinociceptive effect of 5-Iisa, the effects of 5-Fisa and 5-Clisa were reverted by all antagonists used (L-NAME [inhibitor of the nitric oxide synthase enzymes, 3 mg/kg, po]; mecamylamine [nicotinic antagonist, 2 mg/kg, ip]; atropine [muscarinic antagonist, 1 mg/kg, ip]; ondansetron [5-HT3 serotoninergic antagonist, 0.5 mg/kg, ip]; yohimbine [alpha-2 adrenergic antagonist, 2 mg/kg, ip]). On the other hand, 5-Meisa's antinociceptive effect was inhibited by pretreatment of mice with naloxone, atropine or ondansetron (Fig. 6). We also evaluated the effects of antagonists in the model of formalin-induced licking response. Atropine did not demonstrate any significant effect versus either substance in either phase (1st or 2nd). Naloxone was the only antagonist that reverted the inhibitory effect of all the substances. Although L-NAME, mecamylamine and ondansetron reverted the 2nd phase of the licking response to all substances, the effects of these three antagonists in the 1st phase were variable (Fig. 7). In view of the significant results obtained in several models we also decided to further evaluate a possible toxic effect. Even when a dose of 150 mg/kg was used any kind of behavioural or histological alteration was observed (data not shown) suggesting that, at the doses used, these substances do not induce visual signs of toxicity. 4. Discussion
Fig. 2. Effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa on formalin-induced licking behaviour. Mice were pretreated with one of the substances, acetylsalicylic acid (ASA, 200 mg/kg), morphine (5 mg/kg) or vehicle. The results are presented as the mean ± SD (n = 6–10) of the time that the animal spent licking the formalin-injected paw. Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P b 0.05 compared to vehicletreated mice; #P b 0.05 compared to the morphine-treated group; P b 0.05 compared to the ASA-treated group.
In the present study we investigated the antinociceptive effects of four 3-(2-oxopropyl)-3-hydroxy-2-oxindoles using inflammatory and thermal pain models in mice. One interesting finding of this study was that when administered orally, all substances caused inhibition of the formalin-induced nociceptive response, even at doses as low as 0.1 mg/kg. Moreover, these effects were observed in both the 1st and 2nd phases. After subcutaneous injection of formalin an array of stereotyped responses occur that characterise the model as a useful methodology for studying acute and tonic pain. The first phase (or neurogenic) is due to stimulation of nociceptors, whereas the second phase (or inflammatory) is mediated by peripheral and spinal cord sensitisation (Hunskaar and Hole, 1987; Tjølsen et al., 1992). It was also shown that several mediators, such as amino acids, prostaglandin E2, nitric oxide (NO), substance P (SP), serotonin (5-HT) and histamine are increased at the spinal level (Tjølsen et al., 1992; Parada et al., 2001). Our results indicate that the four substances may act through nociceptors (because inhibition of the first phase was observed) or through altered sensitisation of the peripheral or spinal cord. It is also possible that a reduction in some of the inflammatory mediators liberated in the paw occurs. The administration of L-NAME (a NO synthase inhibitor) significantly reversed the antinociceptive effect induced by the oxindoles in the formalininduced licking behaviour. These data support the idea that the substances produce their antinociceptive effects through the involvement of nitric oxide pathways, as is the case for some anti-inflammatory drugs (Aguirre-Bañuelos and Lozano-Cuenca et al., 2005). Ondansetron (a 5-HT3 receptor antagonist) also reversed the effects of all oxindoles suggesting the involvement of the 5-HT3 receptor subtype pathway in
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Fig. 3. Effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa in the tail flick model in mice. Mice were pretreated with one of the substances, morphine (5 mg/kg) or vehicle. The results are presented as the mean ± SD (n = 6–10) of the increase in baseline. Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P b 0.05 when comparing treated mice to vehicletreated mice. Where no error bars are shown it is because they are smaller than the symbol.
the antinociceptive action of the substances. This finding corroborates other data showing that 5-HT receptor antagonists reduce the second phase of formalin-induced aversive responses without affecting the first one (Oyama et al., 2002; Bardin et al., 2001). Putative inflammatory mediators such as glutamate and capsaicin are also able to induce nociceptive pain responses after injection into the paw (Carstens, 1997; Hong and Abott, 1994). Based on this evidence, we sought to determine whether the four compounds inhibit the nociceptive response caused by intraplantar injection of glutamate or capsaicin into the mouse hindpaw. Our data demonstrate that the compounds produced a significant inhibition of the licking response
Fig. 4. Effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa in the tail flick model in mice. Mice were pretreated with one of the substances, morphine (5 mg/kg) or vehicle. The results are presented as the mean ± SD (n = 6–10) of the area under the curve (AUC). Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P b 0.05 when comparing treated mice to vehicle-treated mice and #P b 0.05 when comparing substance-treated mice with morphine-treated animals.
Fig. 5. Effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa on capsaicin (A) and glutamate (B) induced licking behaviour. Mice were orally pretreated with each substance or vehicle 60 min before capsaicin or glutamate injection into their paw. The results are presented as the mean ± SD (n = 6–10) of the time that the animal spent licking the capsaicin- or glutamate-injected paw. Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P b 0.05 when comparing treated mice to vehicle-treated animals.
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Fig. 6. Effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa in the tail flick model. Mice were pretreated with naloxone (opioid antagonist, 5 mg/kg, ip), mecamylamine (nicotinic antagonist, 2 mg/kg, ip), atropine (muscarinic antagonist, 1 mg/kg, ip), ondansetron (5-HT3 serotoninergic antagonist, 0.5 mg/kg, ip), yohimbine (alpha-2 adrenergic antagonist, 2 mg/kg, ip) or L-nitro arginine methyl ester (L-NAME, inhibitor of nitric oxide synthase, 3 mg/kg, ip) 15 min before oral administration of substances (10 mg/kg, po) or vehicle. The results are presented as the mean ± SD (n = 6–10) of the area under the curve (AUC). Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P b 0.05 when comparing treated mice to vehicle-treated mice and #P b 0.05 when comparing antagonist-treated groups with substances-treated animals.
caused by intraplantar injection of both mediators into the hindpaw. This inhibitory effect seems to be more pronounced in animals whose paws were injected with glutamate. Thus, the present results indicate that, at least in part, the antinociceptive action caused by oxindoles
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could be due to an interaction with the glutamatergic system. Capsaicin acts through vanilloid receptor type-1 (TRPV-1) expressed on nociceptive fibres and in the dorsal root ganglion of the spinal cord, trigeminal ganglia and central nervous system (Jancso, 1978; Oyama et al., 2002). The activation of TRPV1 receptors is also mediated by the release of neurotransmitters (i.e., glutamate and substance P), an effect that can participate in nociceptive processing (Afrah et al., 2001; Medvedeva et al., 2008). Glutamate is another neurotransmitter involved in nociceptive transmission, the development and maintenance of a nociceptive response through activation of ionotropic or metabotropic receptors, resulting in nociceptor sensitisation (Aanonsen and Wilcox, 1989; Budai et al., 1995; Carlton, 2001). An intraplantar injection of glutamate also induces the release of excitatory amino acids, PGE2, NO and kinins (Beirith et al., 2002; Sakurada et al., 1998) and promotes the activation of sensitive fibres that induce the release of several substances in the dorsal horn, which also can activate the TRPV1 receptor in the spinal cord (Julius and Basbaum, 2001; Millan, 2002). Our results are in accordance with previous data because the oxindoles reduce the nociceptive effects evoked by both glutamate and capsaicin. These results indicate that the antinociceptive effect of our substances involves, at least in part, the glutamatergic system. In addition to the results obtained in the capsaicin-induced nociception test, it is possible that they could produce antinociception by regulating TRPV1 receptors, thus contributing to the modulation of nociceptive transmission. To assess whether there would be spinal involvement in the spinal effect of oxindoles, we evaluated their antinociceptive effects in the tail flick model. All four substances had effects comparable to morphine with slight differences. The opioid reached its maximal effect at 80 min post-administration and decayed thereafter, whereas oxindoles reached their maximal effect sooner (i.e., 20 or 30 min) and maintained a plateau. It is well known that crosstalk between the adrenergic, opioidergic, nitrergic and serotoninergic pathways occurs at central and peripheral levels. For example, opioid and adrenergic receptors are located at the locus coeruleus and alpha-2 adrenergic receptors are localised on capsaicin-sensitive afferent fibres (Chen et al., 2007; Li and Eisenach, 2001; Fairbanks et al., 2002; Stein et al., 2009). This fact could explain the observation that all the antagonists used were able to reverse the antinociceptive effects of 5-Fisa and 5-Clisa in the tail flick model. It could be that the presence of fluoro or chloro atoms in their structures confers better solubility, facilitating their access to central sites of action. Behavioural alterations such as motor uncoordination and sedation might be misinterpreted as analgesia and produce false positive effects (Tabarelli et al., 2004). In the present study, oral administration of oxindoles did not cause motor impairment as evaluated by either forced locomotion in the rotarod or spontaneous locomotion in the open-field test. Thus, the possibility that the antinociceptive effect of the compounds tested is due to any degree of motor impairment or sedation is very low. We also observed that DMSO per se had any toxic or antiinflammatory/antinociceptive effect. Colucci et al. (2008) had demonstrated that the solvent has an antinociceptive effect when administered by different routes. However in that paper authors used a volume of 0.1 ml DMSO to each animal. In the present study we used a volume of 0.3 μl DMSO per animal (corresponding to the higher dose used, i.e., 10 mg/kg), a volume 333-fold lower than that used in the paper, demonstrating that this volume has any effect. We had previously showed that other analogue of Convolutamydine A, with a bromide atom at position 5, had significant effect in inflammatory (Fernandes et al., 2014) or antinociceptive models (Figueiredo et al., 2013). In those papers the effects were observed with doses ranging from 0.01 to 30 mg/kg. To the formalin-induced licking behaviour the molecule with a 5-Br substituent did not demonstrate any effect at 1st phase, and a significant effect at 2nd phase of the model was observed with 1 or 10 mg/kg doses. In the present work the new substances differ in the substituents. Either iodine, fluoride, chloride or methyl substituents at position 5 increase the activity of the molecule.
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T.B.S. Giorno et al. / Pharmacology, Biochemistry and Behavior 135 (2015) 13–19
Fig. 7. Effects of 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa in the formalin-induced licking model. Mice were pretreated with atropine (muscarinic antagonist, 1 mg/kg, ip), naloxone (opioid antagonist, 5 mg/kg, ip), mecamylamine (nicotinic antagonist, 2 mg/kg, ip), yohimbine (alpha-2 adrenergic antagonist, 2 mg/kg, ip), L-nitro arginine methyl ester (L-NAME, inhibitor of nitric oxide synthase, 3 mg/kg, ip) or ondansetron (5-HT3 serotoninergic antagonist, 0.5 mg/kg, ip), 15 min before oral administration of substances (10 mg/kg, po) or vehicle. The results are presented as the mean ± SD (n = 6–10) of the time that mice spent licking the formalin-injected paw. Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P b 0.05 when comparing treated mice to vehicle-treated mice and #P b 0.05 when comparing antagonist-treated groups with substances-treated animals.
In this regard these substances demonstrate a significant effect even in the 1st phase of formalin-induced licking. All of them also demonstrate an effect in the hot plate model at doses of 0.1 mg/kg. It may be that these substituents confer stability to the molecule or facilitate the interaction between the molecule and the biological target increasing its effects. In conclusion, the data from the present study show that oxindoles exert pronounced systemic antinociception in chemical (formalin, glutamate and capsaicin-induced pain) and spinal (tail flick) models of nociception. In addition, the antinociceptive effects of the oxindoles were antagonised by L-NAME, naloxone, and ondansetron, but not by atropine in the formalin test (Table 1). Thus, these results suggest that oxindoles are able to produce antinociception by the activation of the nitric oxide, opioid or serotoninergic pathways.
Table 1 A summary of the possible mechanism of action to 5-Iisa, 5-Fisa, 5-Clisa and 5-Meisa. Receptor/System involved Opioid
NO
Muscarinic
Nicotinic
α2-adrenergic
5HT3
Substance
F
TF
F
TF
F
TF
F
TF
F
TF
F
TF
5-Iisa 5-Fisa 5-Clisa 5-Meisa
Y Y Y Y
Y Y Y Y
Y Y Y Y
– Y Y –
– – – –
– Y Y Y
– Y Y Y
– Y Y –
– – – –
– Y Y –
Y Y Y Y
– Y Y Y
F, formalin-induced licking; TF, tail flick; –, without effect.
Acknowledgments We would like to thank Mr. Alan Minho for technical assistance and Instituto Vital Brazil (Niterói, Brazil) for donation of mice. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant support and fellowship to PDF), Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ, grant support to PDF), and Conselho de Administração de Pessoal de Ensino Superior (CAPES, fellowship to TBSG and YLLB).
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