Research Article
Involvement of Nitric Oxide and ATP-Sensitive Potassium Channels in the Peripheral Antinoceptive Action of a Tramadol–Dexketoprofen Combination in the Formalin Test
DDR
DRUG DEVELOPMENT RESEARCH 75 : 449–454 (2014)
Mario A. Isiordia-Espinoza,1 Amaury Pozos-Guillén,1* José Pérez-Urizar,2 and Daniel Chavarría-Bolaños1,3 1 Laboratorio de Ciencias Básicas, Facultad de Estomatología, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico 2 Laboratorio de Farmacología, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico 3 Facultad de Odontología, Universidad de Costa Rica, San José, Costa Rica
Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics
Preclinical Research
Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics
Clinical Development Phases I-III Regulatory, Quality, Manufacturing
Postmarketing Phase IV
ABSTRACT Systemic coadministration of tramadol and dexketoprofen can produce antinociceptive synergism in animals. There has been only limited evaluation of this drug combination in the peripheral nervous system in terms of the antinociceptive interaction and its mechanisms. The aim of the present study was to evaluate the peripheral antinociceptive interaction between tramadol and dexketoprofen in the formalin test and the involvement of the nitric oxide (NO)–cyclic guanosine monophosphate pathway and ATP-sensitive K+ channels. Different doses of tramadol or dexketoprofen were administered locally to the formalin-injured mouse paw and the antinociceptive effect evaluated. ED50 values were calculated for both drugs alone and in combination. Coadministration of tramadol and dexketoprofen produced an antinociceptive synergistic interaction during the second phase of the formalin test. Pretreatment with NO antagonists, including l-NG-nitroarginine methyl ester and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1one, or the ATP-sensitive K+ channel antagonist glibenclamide reversed the antinociceptive synergistic effect of the tramadol–dexketoprofen combination, suggesting that NO and ATP-sensitive K+ channels were involved. Drug Dev Res 75 : 449–454, 2014. © 2014 Wiley Periodicals, Inc. Key words: tramadol; dexketoprofen; synergism; formalin test
INTRODUCTION
In moderate to severe pain, it is difficult to obtain effective analgesia using a single drug. As a result, analgesic drug combinations are widely used. Multimodal analgesia is beneficial in the management of different types of both acute and chronic pain [Curatolo and Sveticic, 2002]. Generally, the combined drugs are analgesics with different mechanisms of action that are intended to increase analgesic efficacy while decreasing the doses of the individual agents and thus reducing the © 2014 Wiley Periodicals, Inc.
*Correspondence to: Amaury Pozos-Guillén, Facultad de Estomatología, Universidad Autónoma de San Luis Potosí. Av. Dr. Manuel Nava #2, Zona Universitaria, CP 78290, San Luis Potosí, SLP, Mexico. E-mail: [email protected] Received 17 May 2014; Accepted 01 June 2014 Published online in Wiley Online Library (wileyonlinelibrary .com). DOI: 10.1002/ddr.21180
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adverse effects [Curatolo and Sveticic, 2002; Kehlet and Wilmore, 2002; Pasero, 2003]. The combination of dexketoprofen, a nonsteroidal anti-inflammatory drug, and tramadol, a weak opioid agonist, has been investigated in different animal models of nociception after systemic [Miranda et al., 2009, 2012] and local [Cialdai et al., 2013] administration, with the latter study demonstrating the involvement of the opioid pathways in the local effect of this analgesic combination. However, other pathways have not been extensively evaluated in the peripheral interaction of these drugs. The aim of the present study was to evaluate the peripheral interaction between tramadol and dexketoprofen in the formalin test, assessing the possible role of the nitric oxide (NO) pathway and ATP-sensitive potassium channels in the synergy induced by the tramadol–dexketoprofen combination (TDC).
MATERIALS AND METHODS
Animals Male BALB/c mice (8–9 weeks old, 20–25 g) were housed at 22°C under a 12-h/12-h light/dark cycle. Animals had free access to food and tap water up to the time of the experiment. All experiments were conducted in accordance with the Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals [Zimmerman, 1983], with the study being approved by our local ethics committee.
Drugs Tramadol and dexketoprofen were provided by Stein Laboratories S.A. (San Jose, Costa Rica). LNG-nitroarginine methyl ester (L-NAME), 1H-[1, 2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ), and glibenclamide were obtained from Sigma Chemical Co. (St. Louis, MO, USA). L-NAME was dissolved in sterile saline solution, and ODQ and glibenclamide were dissolved in a 20% dimethyl sulfoxide (DMSO) solution.
Measurement of Nociceptive Activity Nociception was assessed using the formalin test. Mice were placed in clear plastic chambers with a mirror placed at a 45-degree angle to allow an unobstructed view of the paw. The injection was made into the plantar surface of the right hind paw with 30 μL of diluted 3% formalin using a 30-gauge needle. Animals were then returned to the chambers. Nociceptive behavior was quantified as time spent licking the Drug Dev. Res.
injected paw. The total time spent licking after formalin administration, corresponding to the second phase of the assay, was determined to be 15–45 min. Mice were sacrificed in a CO2 chamber at the end of the experiment.
Design Dose–response curves for tramadol- and dexketoprofen-mediated antinociception following local administration (56, 100, 178, and 316 μg/paw for tramadol; 56, 78.6, 100, and 178 μg/paw for dexketoprofen) were assessed, and the doses producing an effect in 50% of the mice (ED50 values) were determined, together with the standard errors of the mean (SEMs) [Tallarida, 2000]. Both drugs were dissolved in 30 μL of 3% formalin. Controls were administered with saline solution in 30 μL of 3% formalin. Once the dose–response curve of each drug was obtained, an experimental ED50 value was determined for each drug. To assess the possible mechanisms of action, the effect of TDC was tested in the presence of LNAME (50 μg/paw), ODQ (50 μg/paw), glibenclamide (100 μg/paw), or vehicle. L-NAME was locally administered with the TDC in 30 μL of 3% formalin. ODQ and glibenclamide were administered in the same manner, with the TDC dissolved in 30 μL of 20% DMSO and 3% formalin. Nociception was assessed immediately afterward. In order to determine whether the drugs acted locally, the highest tested doses of TDC were administered to the left (contralateral) paw 3 min before the 3% formalin was injected into the right paw, and the corresponding effect on nociceptive behavior was assessed. Similarly, antagonists were administered to the left paw before the TDC and 3% formalin were administered to the right paw; nociceptive behavior was then evaluated.
Statistical Analysis Isobolographic analysis and interaction index were used as previously described [Tallarida, 2000; Isiordia-Espinoza et al., 2013]. The theoretical additive ED50 and the experimentally derived ED50 values were evaluated using Student’s t-test. An experimental ED50 value significantly lower than theoretical additive ED50 value was considered to indicate a synergistic interaction between tramadol and dexketoprofen. Mechanisms of action were evaluated by one-way ANOVA followed by the Student–Newman–Keuls test. Statistical significance was considered to be achieved at P < 0.05.
SYNERGY BETWEEN TRAMADOL AND DEXKETOPROFEN
451
RESULTS
Local Antinociceptive Effects of Tramadol, Dexketoprofen, and Their Combination Formalin administration resulted in a common pattern of licking behavior. The nociceptive responses presented with the typical biphasic time courses as follows: an early, short-lasting first period of activity (first phase), followed by a 15-min quiescent period and a second, prolonged tonic phase (second phase). Local administration of tramadol, dexketoprofen, and the TDC produced dose-dependent suppression of the licking response in the second phase of the formalin test, but not in the first. Figure 1 shows the effect of both drugs and their combination. Table 1 depicts the drug doses used in combination.
Isobolographic Analysis and Interaction Index The behavioral responses induced when tramadol and dexketoprofen were administered in combination demonstrated an antinociceptive effect of the synergistic type because the experimental point was below that of the additive line, which was confirmed with an interaction index of less than 1 (Table 2, Fig. 2).
Mechanisms of Action The antinociceptive effect of the TDC was partially reversed by the NO synthesis inhibitor L-NAME, the soluble guanylyl cyclase inhibitor ODQ, and the ATP-sensitive K+ channel antagonist glibenclamide (Fig. 3). All these results were confirmed by the interaction index (Table 2). Contralateral administration of the antagonists did not produce a significant decrease of the antinociceptive effect of the TDC.
DISCUSSION
The present work confirmed the antinociceptive effect of tramadol when locally administered in the formalin test, confirming previous findings demonstrating a dose-dependent effect of tramadol in this same animal pain test [Pozos-Guillén et al., 2006; Isiordia et al., 2010]. The local administration of dexketoprofen produced an antinociceptive effect in the second phase of the formalin test, but not in the first phase. These outcomes confirmed the dose-dependent effect of local dexketoprofen, as reported by Cialdai et al. [2013]. However, in that study, the maximal antinociceptive activity of locally administered dexketoprofen was about 40%, reached using 100 μg/25 μL. In our study, the maximal antinociceptive effect was 74%, which was
Fig. 1. Dose–response curves for the antinociceptive effect of tramadol (A), dexketoprofen (B), and their combination (C) in the second phase of the formalin test.
TABLE 1. Drug Doses Used in Combination (μg/paw) Combination ED50/0.5 ED50/1 ED50/2 ED50/4
Tramadol
Dexketoprofen
132.2 66.1 33.0 16.5
89.0 44.5 22.3 11.1
ED50 = dose at which an effect was observed in 50% of rats.
Drug Dev. Res.
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TABLE 2. Type of Interaction between Tramadol and Dexketoprofen
TDC TDC TDC plus l-NAME (50 μg/paw) TDC plus ODQ (50 μg/paw) TDC plus glibenclamide (100 μg/paw)
Maximal effect*
ED50
Interaction index
Interaction type
71.84 75.86 35.22 45.73 31.49
110.62 ± 5.79 80.87 ± 6.76∧ 751.83 ± 268.49† 288.25 ± 0.62† 1,224.95 ± 520†
1 0.73 6.79 2.60 11.07
Theoretical additivity Synergy Antagonism Antagonism Antagonism
*Percentage of antinociception. ∧ Significantly different (P < 0.05) vs. theoretical additivity ED50. †Significantly different (P < 0.05) vs. experimental ED50. TDC, tramadol–dexketoprofen combination; L-NAME, L-NG-nitroarginine methyl ester; ODQ, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one.
Fig. 2. Isobologram showing the antinociceptive synergistic interaction between tramadol and dexketoprofen in the second phase of the formalin test.
obtained with 178 μg/30 μL (paw). The lack of antinociceptive activity of local dexketoprofen in the first phase of the formalin test was consistent with previous studies [Kolesnikov et al., 2003; Zelcer et al., 2005; Isiordia-Espinoza et al., 2012, 2013]. Systemic interaction between tramadol and dexketoprofen has been previously investigated. Miranda et al. [2009] demonstrated that intragastric administration of a TDC produced a synergistic antinociceptive effect in several animal pain tests. Interestingly, the proportion of the drugs in the TDC could change the type of interaction from synergy to antagonism. Furthermore, Miranda et al. [2012] showed that TDC induced antiexudative and antinociceptive synergism when the TDC was administered at a 1:1 proportion in a murine model of chronic inflammation, suggesting that the combination could be advantageous in the management of musculoskeletal pain in humans. The synergistic interaction in the inhibition of plasma Drug Dev. Res.
Fig. 3. Effect of L-NAME (50 μg/paw) (A), ODQ (50 μg/paw) (B), and glibenclamide (100 μg/paw) (C) on the antinociceptive effect of the TDC in the second phase of the formalin test. Bars are the mean ± SEM for at least six animals. *P < 0.05 versus the combination by one-way ANOVA followed by Student–Newman–Keuls test.
SYNERGY BETWEEN TRAMADOL AND DEXKETOPROFEN
extravasation is remarkable and possesses important therapeutic implications. Specifically, it challenges the artificial division of drugs for arthritis into those that manage symptoms and those that modify the disease. Cialdai et al. [2013] evaluated the effects of oral or intra-articular administration of dexketoprofen and tramadol alone and in combination on nociception in osteoarthritis of the knee induced by intra-articular monosodium iodoacetate and showed that the TDC produced an antinociceptive synergistic effect. It is noteworthy that the synergistic interaction in this work was obtained using lower doses (intra-articular administration; 10 μg of dexketoprofen and 25 μg of tramadol) of both drugs. In our study, local administration of the TDC produced antinociceptive synergy in the second phase of the formalin test. Cialdai et al. [2013] evaluated the participation of opioid receptors in the peripheral antinociception of the TDC in monosodium iodoacetate-induced osteoarthritis in rats and found that naloxone, an opioid receptor antagonist, diminished the effect of the TDC. In this study, several mechanisms of action were analyzed for the TDC. In the present study, the antinociceptive effect of the TDC was prevented by L-NAME [Gibson et al., 1990] and ODQ [Moro et al., 1996], indicating a role for the NO–cyclic guanosine monophosphate (cGMP) pathway at the peripheral level in the formalin test, consistent with previous findings [Lorenzetti and Ferreira, 1996; Granados-Soto et al., 1997; Islas-Cadena et al., 1999; Sachs et al., 2004; Brito et al., 2006; HernándezPacheco et al., 2008]. Likewise, local peripheral administration of glibenclamide, an ATP-sensitive K+ channel blocker [Edwards and Weston, 1993; Mixcoatl-Zecuatl et al., 2006; Hernández-Pacheco et al., 2008], also prevented the antinociceptive effect of the TDC. Taken together, these data suggest that the TDC produces local peripheral antinociception in the formalin test by activation of the NO–cGMP pathway and ATP-sensitive K+ channels. The TDC has been evaluated in three clinical trials. Metscher et al. [2001] performed a clinical study to assess the analgesic efficacy and tolerability of TDC and tramadol in patients with acute lower back pain and found that TDC produced improved analgesia and tolerability. Gómez-Rojas [2013] evaluated the TDC in comparison with the tramadol–ketorolac combination and showed that TDC had a better analgesic effect. Similarly, Ekmekçi et al. [2012] compared the analgesic efficacy of TDC with tramadol for postlaparoscopic cholecystectomy pain treatment and demonstrated that patients receiving TDC had lower visual analog scale scores, increased satisfaction, and decreased opioid consumption when compared with those receiving tramadol alone.
453
In conclusion, the coadministration of tramadol and dexketoprofen produced a synergistic antinociceptive effect in the peripheral nervous system, involving NO and ATP-sensitive K+ channels.
ACKNOWLEDGMENTS
Mario Alberto Isiordia-Espinoza is a PROMEP fellow. Daniel Chavarría-Bolaños is a CONACyTMéxico fellow (#255925). This work was supported partially by grant C14-FAI-04-01.01.
REFERENCES Brito GA, Sachs D, Cunha FQ, Vale ML, Lotufo CM, Ferreira SH, Ribeiro RA. 2006. Peripheral antinociceptive effect of pertussis toxin: activation of the arginine/NO/cGMP/PKG/ATPsensitive K channel pathway. Eur J Neurosci 24:1175–1181. Cialdai C, Giuliani S, Valenti C, Tramontana M, Maggi CA. 2013. Comparison between oral and intra-articular antinociceptive effect of dexketoprofen and tramadol combination in monosodium iodoacetate-induced osteoarthritis in rats. Eur J Pharmacol 714:346–351. Curatolo M, Sveticic G. 2002. Drug combinations in pain treatment: a review of the published evidence and a method for finding the optimal combination. Best Pract Res Clin Anaesthesiol 16:507– 519. Edwards G, Weston AH. 1993. Induction of a glibenclamide-sensitive K-current by modification of a delayed rectifier channel in rat portal vein in insulinoma cells. Br J Pharmacol 110:1280–1281. Ekmekçi P, Kazan-Bengisun Z, Kazbek BK, Özis SE, Tastan H, Süer AH. 2012. The efficacy of adding dexketoprofen trometamol to tramadol with patient controlled analgesia technique in post-laparoscopic cholecystectomy pain treatment. Agri 24:63–68. Gibson A, Mirzazadeh S, Hobbs AJ, Moore PK. 1990. l-NGmonomethyl arginine and l-NG-nitro arginine inhibit nonadrenergic, non-cholinergic relaxation of the mouse anococcygeus muscle. Br J Pharmacol 99:602–606. Gómez-Rojas JP. 2013. Tramadol-ketorolaco versus tramadoldexketoprofeno en pacientes postoperados de prótesis de cadera y rodilla. Rev Mex Anest 36:32–36. Granados-Soto V, Rufino MO, Gomes-Lopes LD, Ferreira SH. 1997. Evidence for the involvement of the nitric oxide–cGMP pathway in the antinociception of morphine in the formalin test. Eur J Pharmacol 340:177–180. Hernández-Pacheco A, Araiza-Saldaña CI, Granados-Soto V, Mixcoatl-Zecuatl T. 2008. Possible participation of the nitric oxide– cyclic GMP–protein kinase G–K+ channels pathway in the peripheral antinociception of melatonin. Eur J Pharmacol 596:70–76. Isiordia M, Pozos-Guillén A, Aguirre-Bañuelos P, Pérez-Urizar J. 2010. Análisis isobolográfico de la combinación analgésica ketorolaco sistémico y tramadol local en el modelo de la formalina en ratones. Rev Mex Cien Farm 41:42–49. Isiordia-Espinoza MA, Terán-Rosales F, Reyes-García G, Granados-Soto V. 2012. Synergism between tramadol and meloxicam in the formalin test involves both opioidermic and serotonergic pathways. Drug Dev Res 73:43–50.
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Kolesnikov YA, Wilson RS, Pasternak GW. 2003. Synergistic interactions between hydrocodone and ibuprofen. Anesth Analg 97:1721–1723. Lorenzetti BB, Ferreira SH. 1996. Activation of the arginine–nitric oxide pathway in primary sensory neurons contributes to dipyrone-induced spinal and peripheral analgesia. Inflamm Res 45:308–311. Metscher B, Kübler U, Jahnel-Kracht H. 2001. Dexketoprofen– trometamol and tramadol in acute lumbago. Fortschr Med Orig 11:147–151. Miranda HF, Puig MM, Romero MA, Prieto JC. 2009. Effects of tramadol and dexketoprofen on analgesia and gastrointestinal transit in mice. Fundam Clin Pharmacol 23:81–88. Miranda HF, Romero MA, Puig MM. 2012. Antinociceptive and anti-exudative synergism between dexketoprofen and tramadol in a model of inflammatory pain in mice. Fundam Clin Pharmacol 26:373–382.
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Pozos-Guillén AJ, Aguirre-Bañuelos P, Arellano-Guerrero A, Castañeda-Hernández G, Hoyo-Vadillo C, Pérez-Urizar J. 2006. Isobolographic analysis of the dual-site synergism in the antinociceptive response of tramadol in the formalin test in rats. Life Sci 79:2275–2282. Sachs D, Cunha FQ, Ferreira SH. 2004. Peripheral analgesic blockade of hypernociception: activation of arginine/NO/cGMP/protein kinase G/ATP-sensitive K+ channel pathway. Proc Natl Acad Sci USA 101:3680–3685. Tallarida RJ. 2000. Drug synergism and dose-effect data analysis. Boca Raton, FL, USA, Chapman Hall/CRC Press. Zelcer S, Kolesnikov Y, Kovalyshyn I, Pasternak DA, Pasternak GW. 2005. Selective potentiation of opioid analgesia by nonsteroidal anti-inflammatory drugs. Brain Res 1040:151–156. Zimmerman M. 1983. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16:109–110.
Involvement of Nitric Oxide and ATP-Sensitive Potassium Channels in the Peripheral Antinoceptive Action of a Tramadol–Dexketoprofen Combination in the Formalin Test
DDR
DRUG DEVELOPMENT RESEARCH 75 : 449–454 (2014)
Mario A. Isiordia-Espinoza,1 Amaury Pozos-Guillén,1* José Pérez-Urizar,2 and Daniel Chavarría-Bolaños1,3 1 Laboratorio de Ciencias Básicas, Facultad de Estomatología, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico 2 Laboratorio de Farmacología, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico 3 Facultad de Odontología, Universidad de Costa Rica, San José, Costa Rica
Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics
Preclinical Research
Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics
Clinical Development Phases I-III Regulatory, Quality, Manufacturing
Postmarketing Phase IV
ABSTRACT Systemic coadministration of tramadol and dexketoprofen can produce antinociceptive synergism in animals. There has been only limited evaluation of this drug combination in the peripheral nervous system in terms of the antinociceptive interaction and its mechanisms. The aim of the present study was to evaluate the peripheral antinociceptive interaction between tramadol and dexketoprofen in the formalin test and the involvement of the nitric oxide (NO)–cyclic guanosine monophosphate pathway and ATP-sensitive K+ channels. Different doses of tramadol or dexketoprofen were administered locally to the formalin-injured mouse paw and the antinociceptive effect evaluated. ED50 values were calculated for both drugs alone and in combination. Coadministration of tramadol and dexketoprofen produced an antinociceptive synergistic interaction during the second phase of the formalin test. Pretreatment with NO antagonists, including l-NG-nitroarginine methyl ester and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1one, or the ATP-sensitive K+ channel antagonist glibenclamide reversed the antinociceptive synergistic effect of the tramadol–dexketoprofen combination, suggesting that NO and ATP-sensitive K+ channels were involved. Drug Dev Res 75 : 449–454, 2014. © 2014 Wiley Periodicals, Inc. Key words: tramadol; dexketoprofen; synergism; formalin test
INTRODUCTION
In moderate to severe pain, it is difficult to obtain effective analgesia using a single drug. As a result, analgesic drug combinations are widely used. Multimodal analgesia is beneficial in the management of different types of both acute and chronic pain [Curatolo and Sveticic, 2002]. Generally, the combined drugs are analgesics with different mechanisms of action that are intended to increase analgesic efficacy while decreasing the doses of the individual agents and thus reducing the © 2014 Wiley Periodicals, Inc.
*Correspondence to: Amaury Pozos-Guillén, Facultad de Estomatología, Universidad Autónoma de San Luis Potosí. Av. Dr. Manuel Nava #2, Zona Universitaria, CP 78290, San Luis Potosí, SLP, Mexico. E-mail: [email protected] Received 17 May 2014; Accepted 01 June 2014 Published online in Wiley Online Library (wileyonlinelibrary .com). DOI: 10.1002/ddr.21180
450
ISIORDIA-ESPINOZA ET AL.
adverse effects [Curatolo and Sveticic, 2002; Kehlet and Wilmore, 2002; Pasero, 2003]. The combination of dexketoprofen, a nonsteroidal anti-inflammatory drug, and tramadol, a weak opioid agonist, has been investigated in different animal models of nociception after systemic [Miranda et al., 2009, 2012] and local [Cialdai et al., 2013] administration, with the latter study demonstrating the involvement of the opioid pathways in the local effect of this analgesic combination. However, other pathways have not been extensively evaluated in the peripheral interaction of these drugs. The aim of the present study was to evaluate the peripheral interaction between tramadol and dexketoprofen in the formalin test, assessing the possible role of the nitric oxide (NO) pathway and ATP-sensitive potassium channels in the synergy induced by the tramadol–dexketoprofen combination (TDC).
MATERIALS AND METHODS
Animals Male BALB/c mice (8–9 weeks old, 20–25 g) were housed at 22°C under a 12-h/12-h light/dark cycle. Animals had free access to food and tap water up to the time of the experiment. All experiments were conducted in accordance with the Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals [Zimmerman, 1983], with the study being approved by our local ethics committee.
Drugs Tramadol and dexketoprofen were provided by Stein Laboratories S.A. (San Jose, Costa Rica). LNG-nitroarginine methyl ester (L-NAME), 1H-[1, 2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ), and glibenclamide were obtained from Sigma Chemical Co. (St. Louis, MO, USA). L-NAME was dissolved in sterile saline solution, and ODQ and glibenclamide were dissolved in a 20% dimethyl sulfoxide (DMSO) solution.
Measurement of Nociceptive Activity Nociception was assessed using the formalin test. Mice were placed in clear plastic chambers with a mirror placed at a 45-degree angle to allow an unobstructed view of the paw. The injection was made into the plantar surface of the right hind paw with 30 μL of diluted 3% formalin using a 30-gauge needle. Animals were then returned to the chambers. Nociceptive behavior was quantified as time spent licking the Drug Dev. Res.
injected paw. The total time spent licking after formalin administration, corresponding to the second phase of the assay, was determined to be 15–45 min. Mice were sacrificed in a CO2 chamber at the end of the experiment.
Design Dose–response curves for tramadol- and dexketoprofen-mediated antinociception following local administration (56, 100, 178, and 316 μg/paw for tramadol; 56, 78.6, 100, and 178 μg/paw for dexketoprofen) were assessed, and the doses producing an effect in 50% of the mice (ED50 values) were determined, together with the standard errors of the mean (SEMs) [Tallarida, 2000]. Both drugs were dissolved in 30 μL of 3% formalin. Controls were administered with saline solution in 30 μL of 3% formalin. Once the dose–response curve of each drug was obtained, an experimental ED50 value was determined for each drug. To assess the possible mechanisms of action, the effect of TDC was tested in the presence of LNAME (50 μg/paw), ODQ (50 μg/paw), glibenclamide (100 μg/paw), or vehicle. L-NAME was locally administered with the TDC in 30 μL of 3% formalin. ODQ and glibenclamide were administered in the same manner, with the TDC dissolved in 30 μL of 20% DMSO and 3% formalin. Nociception was assessed immediately afterward. In order to determine whether the drugs acted locally, the highest tested doses of TDC were administered to the left (contralateral) paw 3 min before the 3% formalin was injected into the right paw, and the corresponding effect on nociceptive behavior was assessed. Similarly, antagonists were administered to the left paw before the TDC and 3% formalin were administered to the right paw; nociceptive behavior was then evaluated.
Statistical Analysis Isobolographic analysis and interaction index were used as previously described [Tallarida, 2000; Isiordia-Espinoza et al., 2013]. The theoretical additive ED50 and the experimentally derived ED50 values were evaluated using Student’s t-test. An experimental ED50 value significantly lower than theoretical additive ED50 value was considered to indicate a synergistic interaction between tramadol and dexketoprofen. Mechanisms of action were evaluated by one-way ANOVA followed by the Student–Newman–Keuls test. Statistical significance was considered to be achieved at P < 0.05.
SYNERGY BETWEEN TRAMADOL AND DEXKETOPROFEN
451
RESULTS
Local Antinociceptive Effects of Tramadol, Dexketoprofen, and Their Combination Formalin administration resulted in a common pattern of licking behavior. The nociceptive responses presented with the typical biphasic time courses as follows: an early, short-lasting first period of activity (first phase), followed by a 15-min quiescent period and a second, prolonged tonic phase (second phase). Local administration of tramadol, dexketoprofen, and the TDC produced dose-dependent suppression of the licking response in the second phase of the formalin test, but not in the first. Figure 1 shows the effect of both drugs and their combination. Table 1 depicts the drug doses used in combination.
Isobolographic Analysis and Interaction Index The behavioral responses induced when tramadol and dexketoprofen were administered in combination demonstrated an antinociceptive effect of the synergistic type because the experimental point was below that of the additive line, which was confirmed with an interaction index of less than 1 (Table 2, Fig. 2).
Mechanisms of Action The antinociceptive effect of the TDC was partially reversed by the NO synthesis inhibitor L-NAME, the soluble guanylyl cyclase inhibitor ODQ, and the ATP-sensitive K+ channel antagonist glibenclamide (Fig. 3). All these results were confirmed by the interaction index (Table 2). Contralateral administration of the antagonists did not produce a significant decrease of the antinociceptive effect of the TDC.
DISCUSSION
The present work confirmed the antinociceptive effect of tramadol when locally administered in the formalin test, confirming previous findings demonstrating a dose-dependent effect of tramadol in this same animal pain test [Pozos-Guillén et al., 2006; Isiordia et al., 2010]. The local administration of dexketoprofen produced an antinociceptive effect in the second phase of the formalin test, but not in the first phase. These outcomes confirmed the dose-dependent effect of local dexketoprofen, as reported by Cialdai et al. [2013]. However, in that study, the maximal antinociceptive activity of locally administered dexketoprofen was about 40%, reached using 100 μg/25 μL. In our study, the maximal antinociceptive effect was 74%, which was
Fig. 1. Dose–response curves for the antinociceptive effect of tramadol (A), dexketoprofen (B), and their combination (C) in the second phase of the formalin test.
TABLE 1. Drug Doses Used in Combination (μg/paw) Combination ED50/0.5 ED50/1 ED50/2 ED50/4
Tramadol
Dexketoprofen
132.2 66.1 33.0 16.5
89.0 44.5 22.3 11.1
ED50 = dose at which an effect was observed in 50% of rats.
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TABLE 2. Type of Interaction between Tramadol and Dexketoprofen
TDC TDC TDC plus l-NAME (50 μg/paw) TDC plus ODQ (50 μg/paw) TDC plus glibenclamide (100 μg/paw)
Maximal effect*
ED50
Interaction index
Interaction type
71.84 75.86 35.22 45.73 31.49
110.62 ± 5.79 80.87 ± 6.76∧ 751.83 ± 268.49† 288.25 ± 0.62† 1,224.95 ± 520†
1 0.73 6.79 2.60 11.07
Theoretical additivity Synergy Antagonism Antagonism Antagonism
*Percentage of antinociception. ∧ Significantly different (P < 0.05) vs. theoretical additivity ED50. †Significantly different (P < 0.05) vs. experimental ED50. TDC, tramadol–dexketoprofen combination; L-NAME, L-NG-nitroarginine methyl ester; ODQ, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one.
Fig. 2. Isobologram showing the antinociceptive synergistic interaction between tramadol and dexketoprofen in the second phase of the formalin test.
obtained with 178 μg/30 μL (paw). The lack of antinociceptive activity of local dexketoprofen in the first phase of the formalin test was consistent with previous studies [Kolesnikov et al., 2003; Zelcer et al., 2005; Isiordia-Espinoza et al., 2012, 2013]. Systemic interaction between tramadol and dexketoprofen has been previously investigated. Miranda et al. [2009] demonstrated that intragastric administration of a TDC produced a synergistic antinociceptive effect in several animal pain tests. Interestingly, the proportion of the drugs in the TDC could change the type of interaction from synergy to antagonism. Furthermore, Miranda et al. [2012] showed that TDC induced antiexudative and antinociceptive synergism when the TDC was administered at a 1:1 proportion in a murine model of chronic inflammation, suggesting that the combination could be advantageous in the management of musculoskeletal pain in humans. The synergistic interaction in the inhibition of plasma Drug Dev. Res.
Fig. 3. Effect of L-NAME (50 μg/paw) (A), ODQ (50 μg/paw) (B), and glibenclamide (100 μg/paw) (C) on the antinociceptive effect of the TDC in the second phase of the formalin test. Bars are the mean ± SEM for at least six animals. *P < 0.05 versus the combination by one-way ANOVA followed by Student–Newman–Keuls test.
SYNERGY BETWEEN TRAMADOL AND DEXKETOPROFEN
extravasation is remarkable and possesses important therapeutic implications. Specifically, it challenges the artificial division of drugs for arthritis into those that manage symptoms and those that modify the disease. Cialdai et al. [2013] evaluated the effects of oral or intra-articular administration of dexketoprofen and tramadol alone and in combination on nociception in osteoarthritis of the knee induced by intra-articular monosodium iodoacetate and showed that the TDC produced an antinociceptive synergistic effect. It is noteworthy that the synergistic interaction in this work was obtained using lower doses (intra-articular administration; 10 μg of dexketoprofen and 25 μg of tramadol) of both drugs. In our study, local administration of the TDC produced antinociceptive synergy in the second phase of the formalin test. Cialdai et al. [2013] evaluated the participation of opioid receptors in the peripheral antinociception of the TDC in monosodium iodoacetate-induced osteoarthritis in rats and found that naloxone, an opioid receptor antagonist, diminished the effect of the TDC. In this study, several mechanisms of action were analyzed for the TDC. In the present study, the antinociceptive effect of the TDC was prevented by L-NAME [Gibson et al., 1990] and ODQ [Moro et al., 1996], indicating a role for the NO–cyclic guanosine monophosphate (cGMP) pathway at the peripheral level in the formalin test, consistent with previous findings [Lorenzetti and Ferreira, 1996; Granados-Soto et al., 1997; Islas-Cadena et al., 1999; Sachs et al., 2004; Brito et al., 2006; HernándezPacheco et al., 2008]. Likewise, local peripheral administration of glibenclamide, an ATP-sensitive K+ channel blocker [Edwards and Weston, 1993; Mixcoatl-Zecuatl et al., 2006; Hernández-Pacheco et al., 2008], also prevented the antinociceptive effect of the TDC. Taken together, these data suggest that the TDC produces local peripheral antinociception in the formalin test by activation of the NO–cGMP pathway and ATP-sensitive K+ channels. The TDC has been evaluated in three clinical trials. Metscher et al. [2001] performed a clinical study to assess the analgesic efficacy and tolerability of TDC and tramadol in patients with acute lower back pain and found that TDC produced improved analgesia and tolerability. Gómez-Rojas [2013] evaluated the TDC in comparison with the tramadol–ketorolac combination and showed that TDC had a better analgesic effect. Similarly, Ekmekçi et al. [2012] compared the analgesic efficacy of TDC with tramadol for postlaparoscopic cholecystectomy pain treatment and demonstrated that patients receiving TDC had lower visual analog scale scores, increased satisfaction, and decreased opioid consumption when compared with those receiving tramadol alone.
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In conclusion, the coadministration of tramadol and dexketoprofen produced a synergistic antinociceptive effect in the peripheral nervous system, involving NO and ATP-sensitive K+ channels.
ACKNOWLEDGMENTS
Mario Alberto Isiordia-Espinoza is a PROMEP fellow. Daniel Chavarría-Bolaños is a CONACyTMéxico fellow (#255925). This work was supported partially by grant C14-FAI-04-01.01.
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