DDR
DRUG DEVELOPMENT RESEARCH 75 : 224–230 (2014)
Research Article
Peripheral Synergism between Tramadol and Ibuprofen in the Formalin Test Daniel Chavarria-Bolaños,1,2 José Perez-Urizar,3 Christian Grandfils,4 and Amaury Pozos-Guillén5* 1 Doctorado Institucional en Ingeniería y Ciencia de Materiales, Universidad Autónoma San Luis Potosí, San Luis Potosi, Mexico 2 Pharmaceutical and Biomedical Sciences PhD Program, University of Liege, Liege, Belgium 3 Laboratorio de Farmacología, Facultad de Ciencias Químicas, Universidad Autónoma San Luis Potosí, San Luis Potosi, Mexico 4 Interfacultary Centre of Biomaterials, University of Liege, Liege, Belgium 5 Laboratorio de Ciencias Básicas, Facultad de Estomatología, Universidad Autónoma San Luis Potosí, San Luis Potosi, Mexico
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 Analgesics with different mechanisms of action can be combined in order to obtain pharmacological synergism, employing lower doses of each agent, thus diminishing side effects. For instance, an atypical dual analgesic such as tramadol (TMD) and a nonsteroidal anti-inflammatory drug such as ibuprofen (IBU) are good candidates to be evaluated when combined and applied peripherally. The present study was conducted to evaluate possible local synergism between TMD and IBU when combined peripherally using the formalin test in rats. The effects of the individual analgesics and their combinations were evaluated simultaneously using a 5% formalin dilution. Dose–effect curves were determined for TMD (50–400 μg/paw) and IBU (1–100 μg/paw). Experimental effective doses were evaluated and isobolographic analyses were constructed to evaluate TMD-IBU combination synergism. Both drugs produced a dose-dependent analgesic effect when applied separately. Isobolographic analysis showed synergism during phase 1 (0–10 min) and phase 2 (15–60 min) when compared with theoretical doses (P < 0.05), with interaction indexes of 0.06 and 0.09, respectively. The present information supports the peripheral analgesic effect of TMD and IBU, especially when combined at appropriate doses. Drug Dev Res 75 : 224–230, 2014. © 2014 Wiley Periodicals, Inc. Key words: tramadol; ibuprofen; formalin test; multimodal analgesia
INTRODUCTION
Different alternatives are available to control pain employing analgesic drugs. For instance, the concept of combining molecules with different mechanisms of action is very attractive [Curatolo and Sveticic, 2002]. Analgesic combinations under the concept of “multimodal analgesia” [Buvanendran and Kroin, 2009; Young and Buvanendran, 2012] offer the possibility of obtaining a greater clinical effect by using lower doses of individual compounds together, while diminishing © 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, San Luis Potosí, SLP CP 78290, Mexico. E-mail: [email protected] Received 7 March 2014; Accepted 9 April 2014 Published online in Wiley Online Library (wileyonlinelibrary .com). DOI: 10.1002/ddr.21175
TRAMADOL–IBUPROFEN COMBINATION
possible side effects [Tallarida, 2001]. A different approach to increasing pharmacological analgesia does not involve the pharmacodynamic profiles of the analgesics involved, but their pharmacokinetics. Analgesics are usually applied systemically before they can reach the desired peripheral action site [Stein and Lang, 2009; Komatsu and Sakurada, 2012; Ceccheti et al., 2014]. More recently, efforts have been applied to exploring local, sustained-release forms applied directly to the site where pain originates [Sawynok, 2003]. While avoiding possible systemic adverse effects, this strategy also decreases any possible interference with other concomitant pharmacological treatments already taken by the patient [Barkin, 2012; Rudin, 2013]. Tramadol (TMD) is a commonly used synthetic, centrally acting analgesic [Scott and Perry, 2000]. Although the mechanism of action is unclear [Yalcin and Aksu, 2005], this drug is a weak μ-opioid receptor (MOR) agonist and a monoamine (norepinephrine and serotonin) uptake inhibitor [Grond and Sablotzki, 2004]. However, the majority of information supporting both mechanisms is based on systemic administration models, thus involving central pharmacological sites [Vahabi et al., 2011]. Ibuprofen (IBU) is a nonsteroidal anti-inflammatory drug (NSAID), acting via cyclooxygenase (COX) inhibition centrally and peripherally. In addition to its main mechanism of decreasing prostaglandin levels, other actions have been ascribed to it, for example attenuation of sodium channel activation and a decrease in the levels of various inflammatory mediators (such as leukotrienes, nitric oxide, or transcription factors). All of these effects are potentially complementary and beneficial in clinical practice, especially when the drug is used peripherally [Rainsford, 2009]. While until now these two analgesics have been mainly considered independently, it was interesting to explore a possible pharmacological synergism between both when they were applied directly to the source of the pain. Thus, the aim of this study was to evaluate the peripheral synergism of a TMD-IBU combination using the formalin test in rats. MATERIALS AND METHODS
Animals Male Wistar rats aged 6–8 weeks (weight range 150–200 g) were chosen for this study. Rats were housed at 22°C with a 12 h/12 h light/dark cycle and had free access to food and tap water up to the time of the experiment. Each animal was used only once and sacrificed at the end of the test by an overdose of anesthetic. All experiments were conducted in accordance with the Guidelines on Ethical Standards for Investiga-
225
tion of Experimental Pain in Animals [Zimmerman, 1983]. The study was also approved by our institutional ethics committee. Drugs IBU and TMD were used without excipients or vehicles (Stein Laboratories, Cartago, Costa Rica). To evaluate local synergism, both drugs were dissolved in dimethyl sulfoxide (DMSO) 8% (Sigma-Aldrich Co., St. Louis, MO), employing staggered dilutions and ultrasonic homogenization to avoid crystallization and/or precipitate formation. Depending on the group assigned, each rat was injected with fixed doses of the drug combinations by intraplantar (i.p.) route. The total volume of injection into the rat paw with the formalin solution or combined with drugs was 50 μL. Control groups of rats received only DMSO 8%. Measurement of Nociceptive Response Nociceptive response was assessed using the formalin test. Rats were placed in transparent plastic cylindrical chambers with a mirror placed at a 45° angle to allow an unobstructed view of the paws. They were injected in the dorsal surface of the hind paw with 50 μL of dilute formalin (5%) alone or mixed with the corresponding concentration of analgesics using a 30 g needle and observed for nociceptive behavior immediately after injection. Nociceptive behavior was quantified as the number of flinches of the injected paws during 1-min periods every 5 min up to 60 min after injection. Time courses of antinociceptive response to individual drugs and combinations were constructed by plotting the mean number of flinches as a function of time. Dose–response data were quantified as the percentage of reduction in flinches relative to the control total in each phase (first phase: 0–10 min; second phase: 15–60 min) of the formalin test. The percentage of antinociception was calculated according to the following equation:
% Antinociception = ((Control − Drug ) Control ) × 100, where “Control” and “Drug” refer to the number of flinches elicited by formalin plus saline and formalin plus drug, respectively. Rats were sacrificed by an overdose of anesthetic at the end of the experiment. Experimental Design Dose–response curves of TMD-IBU-mediated antinociception following local administration of each Drug Dev. Res.
CHAVARRIA-BOLAÑOS ET AL.
Data Analysis and Statistics Results are presented as the mean ± SEM for six animals per group. Dose–response curves were constructed and the experimental points fitted using leastsquares linear regression. In order to evaluate the synergism between both drugs, isobolograms were constructed by using the individual ED50 values obtained. We determined the nature of the interaction between TMD and IBU by isobolographic analysis. This technique assumes that the combination of drugs is made from equipotent doses of the individual drugs; thus, the theoretically additive dose combination was calculated and compared with the experimentally derived one. Such a comparison was carried out by using Student’s t-test, and a difference at the P < 0.05 level was considered as indicating a significant synergistic interaction between TMD and IBU in the combination. In addition, to describe the magnitude of the interaction, the interaction index (γ) was calculated: ([ED50 dose in combination of drug 1]/[ED50 value for drug 1 given alone]) + ([ED50 dose in combination of drug 2]/[ED50 value for drug 2 given alone]). A value near 1 indicates additive interaction, a value >1 implies an antagonistic interaction, and a value
DRUG DEVELOPMENT RESEARCH 75 : 224–230 (2014)
Research Article
Peripheral Synergism between Tramadol and Ibuprofen in the Formalin Test Daniel Chavarria-Bolaños,1,2 José Perez-Urizar,3 Christian Grandfils,4 and Amaury Pozos-Guillén5* 1 Doctorado Institucional en Ingeniería y Ciencia de Materiales, Universidad Autónoma San Luis Potosí, San Luis Potosi, Mexico 2 Pharmaceutical and Biomedical Sciences PhD Program, University of Liege, Liege, Belgium 3 Laboratorio de Farmacología, Facultad de Ciencias Químicas, Universidad Autónoma San Luis Potosí, San Luis Potosi, Mexico 4 Interfacultary Centre of Biomaterials, University of Liege, Liege, Belgium 5 Laboratorio de Ciencias Básicas, Facultad de Estomatología, Universidad Autónoma San Luis Potosí, San Luis Potosi, Mexico
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 Analgesics with different mechanisms of action can be combined in order to obtain pharmacological synergism, employing lower doses of each agent, thus diminishing side effects. For instance, an atypical dual analgesic such as tramadol (TMD) and a nonsteroidal anti-inflammatory drug such as ibuprofen (IBU) are good candidates to be evaluated when combined and applied peripherally. The present study was conducted to evaluate possible local synergism between TMD and IBU when combined peripherally using the formalin test in rats. The effects of the individual analgesics and their combinations were evaluated simultaneously using a 5% formalin dilution. Dose–effect curves were determined for TMD (50–400 μg/paw) and IBU (1–100 μg/paw). Experimental effective doses were evaluated and isobolographic analyses were constructed to evaluate TMD-IBU combination synergism. Both drugs produced a dose-dependent analgesic effect when applied separately. Isobolographic analysis showed synergism during phase 1 (0–10 min) and phase 2 (15–60 min) when compared with theoretical doses (P < 0.05), with interaction indexes of 0.06 and 0.09, respectively. The present information supports the peripheral analgesic effect of TMD and IBU, especially when combined at appropriate doses. Drug Dev Res 75 : 224–230, 2014. © 2014 Wiley Periodicals, Inc. Key words: tramadol; ibuprofen; formalin test; multimodal analgesia
INTRODUCTION
Different alternatives are available to control pain employing analgesic drugs. For instance, the concept of combining molecules with different mechanisms of action is very attractive [Curatolo and Sveticic, 2002]. Analgesic combinations under the concept of “multimodal analgesia” [Buvanendran and Kroin, 2009; Young and Buvanendran, 2012] offer the possibility of obtaining a greater clinical effect by using lower doses of individual compounds together, while diminishing © 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, San Luis Potosí, SLP CP 78290, Mexico. E-mail: [email protected] Received 7 March 2014; Accepted 9 April 2014 Published online in Wiley Online Library (wileyonlinelibrary .com). DOI: 10.1002/ddr.21175
TRAMADOL–IBUPROFEN COMBINATION
possible side effects [Tallarida, 2001]. A different approach to increasing pharmacological analgesia does not involve the pharmacodynamic profiles of the analgesics involved, but their pharmacokinetics. Analgesics are usually applied systemically before they can reach the desired peripheral action site [Stein and Lang, 2009; Komatsu and Sakurada, 2012; Ceccheti et al., 2014]. More recently, efforts have been applied to exploring local, sustained-release forms applied directly to the site where pain originates [Sawynok, 2003]. While avoiding possible systemic adverse effects, this strategy also decreases any possible interference with other concomitant pharmacological treatments already taken by the patient [Barkin, 2012; Rudin, 2013]. Tramadol (TMD) is a commonly used synthetic, centrally acting analgesic [Scott and Perry, 2000]. Although the mechanism of action is unclear [Yalcin and Aksu, 2005], this drug is a weak μ-opioid receptor (MOR) agonist and a monoamine (norepinephrine and serotonin) uptake inhibitor [Grond and Sablotzki, 2004]. However, the majority of information supporting both mechanisms is based on systemic administration models, thus involving central pharmacological sites [Vahabi et al., 2011]. Ibuprofen (IBU) is a nonsteroidal anti-inflammatory drug (NSAID), acting via cyclooxygenase (COX) inhibition centrally and peripherally. In addition to its main mechanism of decreasing prostaglandin levels, other actions have been ascribed to it, for example attenuation of sodium channel activation and a decrease in the levels of various inflammatory mediators (such as leukotrienes, nitric oxide, or transcription factors). All of these effects are potentially complementary and beneficial in clinical practice, especially when the drug is used peripherally [Rainsford, 2009]. While until now these two analgesics have been mainly considered independently, it was interesting to explore a possible pharmacological synergism between both when they were applied directly to the source of the pain. Thus, the aim of this study was to evaluate the peripheral synergism of a TMD-IBU combination using the formalin test in rats. MATERIALS AND METHODS
Animals Male Wistar rats aged 6–8 weeks (weight range 150–200 g) were chosen for this study. Rats were housed at 22°C with a 12 h/12 h light/dark cycle and had free access to food and tap water up to the time of the experiment. Each animal was used only once and sacrificed at the end of the test by an overdose of anesthetic. All experiments were conducted in accordance with the Guidelines on Ethical Standards for Investiga-
225
tion of Experimental Pain in Animals [Zimmerman, 1983]. The study was also approved by our institutional ethics committee. Drugs IBU and TMD were used without excipients or vehicles (Stein Laboratories, Cartago, Costa Rica). To evaluate local synergism, both drugs were dissolved in dimethyl sulfoxide (DMSO) 8% (Sigma-Aldrich Co., St. Louis, MO), employing staggered dilutions and ultrasonic homogenization to avoid crystallization and/or precipitate formation. Depending on the group assigned, each rat was injected with fixed doses of the drug combinations by intraplantar (i.p.) route. The total volume of injection into the rat paw with the formalin solution or combined with drugs was 50 μL. Control groups of rats received only DMSO 8%. Measurement of Nociceptive Response Nociceptive response was assessed using the formalin test. Rats were placed in transparent plastic cylindrical chambers with a mirror placed at a 45° angle to allow an unobstructed view of the paws. They were injected in the dorsal surface of the hind paw with 50 μL of dilute formalin (5%) alone or mixed with the corresponding concentration of analgesics using a 30 g needle and observed for nociceptive behavior immediately after injection. Nociceptive behavior was quantified as the number of flinches of the injected paws during 1-min periods every 5 min up to 60 min after injection. Time courses of antinociceptive response to individual drugs and combinations were constructed by plotting the mean number of flinches as a function of time. Dose–response data were quantified as the percentage of reduction in flinches relative to the control total in each phase (first phase: 0–10 min; second phase: 15–60 min) of the formalin test. The percentage of antinociception was calculated according to the following equation:
% Antinociception = ((Control − Drug ) Control ) × 100, where “Control” and “Drug” refer to the number of flinches elicited by formalin plus saline and formalin plus drug, respectively. Rats were sacrificed by an overdose of anesthetic at the end of the experiment. Experimental Design Dose–response curves of TMD-IBU-mediated antinociception following local administration of each Drug Dev. Res.
CHAVARRIA-BOLAÑOS ET AL.
Data Analysis and Statistics Results are presented as the mean ± SEM for six animals per group. Dose–response curves were constructed and the experimental points fitted using leastsquares linear regression. In order to evaluate the synergism between both drugs, isobolograms were constructed by using the individual ED50 values obtained. We determined the nature of the interaction between TMD and IBU by isobolographic analysis. This technique assumes that the combination of drugs is made from equipotent doses of the individual drugs; thus, the theoretically additive dose combination was calculated and compared with the experimentally derived one. Such a comparison was carried out by using Student’s t-test, and a difference at the P < 0.05 level was considered as indicating a significant synergistic interaction between TMD and IBU in the combination. In addition, to describe the magnitude of the interaction, the interaction index (γ) was calculated: ([ED50 dose in combination of drug 1]/[ED50 value for drug 1 given alone]) + ([ED50 dose in combination of drug 2]/[ED50 value for drug 2 given alone]). A value near 1 indicates additive interaction, a value >1 implies an antagonistic interaction, and a value
