Pain 121 (2006) 22–28 www.elsevier.com/locate/pain Synergism between paracetamol and nonsteroidal anti-inflammatory drugs in experimental acute pain Hugo F. Miranda a,*, Margarita M. Puig b, Juan Carlos Prieto a, Gianni Pinardi a a Pharmacology Program, ICBM, Faculty of Medicine, University of Chile, Clasificador 70.000, Santiago 7, Chile b Department of Anesthesiology, Hospital del Mar, Paseo Marı´timo 25, 08003 Barcelona, Spain Received 9 June 2005; received in revised form 3 November 2005; accepted 21 November 2005 Abstract The antinociception induced by the intraperitoneal coadministration of combinations of paracetamol with the nonsteroidal anti- inflammatory drugs (NSAIDs) diclofenac, ibuprofen, ketoprofen, meloxicam, metamizol, naproxen, nimesulide, parecoxib and pir- oxicam was studied by isobolographic analysis in the acetic acid abdominal constriction test of mice (writhing test). The effective dose that produced 50% antinociception (ED50) was calculated from the log dose–response curves of fixed ratio combinations of paracetamol with each NSAID. By isobolographic analysis, this ED50 was compared to the theoretical additive ED50 calculated from the ED50 of paracetamol and of each NSAID alone obtained from ED50 dose–response curves. As shown by isobolographic analysis, all the combinations were synergistic, the experimental ED50s being significantly smaller than the theoretically calculated ED50s. The results of this study demonstrate potent interactions between paracetamol and NSAIDs and validate the clinical use of combinations of these drugs in the treatment of pain conditions. Ó 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Antinociception; NSAIDs; Paracetamol; Synergy; Abdominal constriction test; Writhing test; Isobologram 1. Introduction The antinociceptive action of nonsteroidal anti-in- flammatory drugs (NSAIDs) is primarily due to the The combination of analgesics of proven efficacy is a inhibition of prostaglandin biosynthesis through the strategy intended to achieve one or more therapeutic inhibition of cyclooxygenase enzymes: COX-1 (constitu- goals, such as facilitating patient compliance, simplifying tive) and COX-2 (inducible in inflammatory processes), prescribing, improving efficacy without increasing even if alternative mechanisms have to be considered adverse effects or decreasing adverse effects without loss (Mitchell and Warner, 1999; Smith et al., 2000; Miranda of efficacy (Raffa, 2001; Hyllested et al., 2002). In certain et al., 2002; Warner and Mitchell, 2004). However, the cases, the coadministration of antinociceptive agents absolute separation between the physiological and path- results in synergistic effects and the doses of the individ- ological roles of COX-1 and COX-2 is becoming less ual drugs can be substantially reduced (Maves et al., tenable and indeed their activities overlap to a consider- 1994; Salazar et al., 1995; Fairbanks and Wilcox, 1999; able degree (Wallace, 1999). On the other hand, the Kolesnikov et al., 2002; Miranda and Pinardi, 2004). mechanism of action of one of the most widely used analgesics, paracetamol or acetaminophen, remains largely unknown and at most the drug is considered to be an atypical NSAID, since it is a weak inhibitor of * Corresponding author. Tel.: +56 2 678 6237; fax: +56 2 737 2783. COXs (Botting, 2003). The following mechanisms have E-mail address: [email protected] (H.F. Miranda). been postulated to explain paracetamol-induced 0304-3959/$20.00 Ó 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2005.11.012 H.F. Miranda et al. / Pain 121 (2006) 22–28 23 analgesia: selective inhibition of cyclooxygenase activity 2.3. Control animals in the CNS, interaction with spinal 5-HT3 receptors, interference with spinal substance P receptors or Physiological salt solution (10 mL/kg) was injected intra- inhibition of neurons excited by substance P, activation peritoneally (i.p.) or given orally and control animals were of suprasegmental descending inhibitory pathways, run interspersed concurrently with the drug treatments, which prevented the controls being run on a single group of mice at increase in pituitary b-endorphin secretion, direct effects one time (19.7 ± 0.28, n = 92 i.p.; 20.1 ± 0.33, n = 12 p.o.). on neuronal membrane potentials (Bannwarth et al., 1995; Pelissier et al., 1995; Pini et al., 1996; Raffa and 2.4. Protocol Codd, 1996; Breivik et al., 1999). NSAIDs and paracetamol are drugs widely used to Dose–response curves for the antinociceptive effect of dic- treat moderate to mild pain, but they are often inad- lofenac, ibuprofen, ketoprofen, meloxicam, metamizol, equate against severe pain. Clinical studies on patients naproxen, nimesulide, paracetamol, parecoxib, and piroxicam with musculoskeletal conditions, dental pain or post- were obtained using at least six animals at each of at least operative pain have shown that combinations of para- four doses. The drugs were injected intraperitoneally. Dose– cetamol and NSAIDs may provide additive pain-relief response curves were also obtained by the oral administration (Altman, 2004). However, a detailed evaluation of dif- of paracetamol and diclofenac. A least-squares linear regres- sion analysis of the log dose–response curves allowed the cal- ferent combinations of paracetamol with commonly culation of the dose that produced 50% of antinociception used NSAIDs has not been reported. The purpose (ED50) for each drug. A dose–response curve was also of the present study was to assess the nature of the obtained by the intraperitoneal coadministration of paraceta- interaction between the systemic administration of mol with each NSAID in fixed ratio combinations of frac- combinations of paracetamol and several NSAIDs in tions of their respective ED50 values: 1/2, 1/4, 1/8, 1/16 a rodent model of visceral pain. A broad number of (ratio values given in Table 2). A dose–response curve for clinical relevant NSAIDs are included because they the combination of paracetamol and diclofenac administered are the most frequently used agents for the treatment orally by gavage was also obtained with the same scheme. of pain (Møiniche et al., 2003; Rømsing and Møini- Isobolographic analysis was used to determine drug interac- che, 2004). tions. The method has been described previously in detail (Miranda et al., 2002). Supra-additivity or synergistic effect is defined as the effect of a drug combination that is higher 2. Materials and methods and statistically different (ED50 significantly lower) than the theoretically calculated equieffect of a drug combination with 2.1. Animals the same proportions. If the ED50s are not statistically differ- ent, the effect of the combination is additive and additivity Male CF-1 mice (28–30 g), housed on a 12 h light–dark means that each constituent contributes with its own potency cycle at 22 ± 2 °C and with access to food and water ad libi- to the total effect. The interaction index was calculated as tum, were used. Experiments were performed in accordance experimental ED50/theoretical ED50. If the value is close to with current Guidelines for The Care of Laboratory Animals 1, the interaction is additive. Values lower than 1 are an indi- and Ethical Guidelines for investigation of experimental pain cation of the magnitude of supra-additive or synergistic inter- approved by the Animal Care and Use Committee of the Fac- actions and values higher than 1 correspond to sub-additive ulty of Medicine, University of Chile. Animals were acclima- or antagonistic interactions (Tallarida, 2001). tized to the laboratory for at least 2 h before testing, were used only once during the protocol and were killed by cervical 2.5. Drugs dislocation immediately after the algesiometric test. The num- ber of animals was kept at a minimum compatible with consis- The drugs were freshly dissolved in a physiological salt solu- tent effects of the drug treatments. tion. Paracetamol was provided by Bristol-Myers-Squibb, France. The NSAIDs were provided by local pharmaceutical 2.2. Measurement of analgesic activity companies: diclofenac by Novartis Chile S.A., ibuprofen by Laboratorio Chile S.A., ketoprofen by Rhone-Poulenc Ror- Analgesic activity was assessed by the acetic acid abdominal er, meloxicam and naproxen by Laboratorios Saval S.A., constriction test (writhing test), a chemical visceral pain model metamizol by Sanderson S.A., nimesulide by Gru¨nenthal (Hayashi and Takemori, 1971). Mice were injected i.p. with Chilena Ltda, parecoxib and piroxicam by Pfizer Chile. 10 mL/kg of 0.6% acetic acid solution 30 min after the i.p. Doses were expressed on the basis of the salts. administration of the drugs, a time at which preliminary exper- iments showed occurrence of the maximum effect. A writhe is 2.6. Statistical analysis characterized by a wave of contraction of the abdominal mus- culature followed by the extension of the hind limbs. The num- Results are presented as means ± SEM or as ED50 values ber of writhes in a 5 min period was counted, starting 5 min with 95% confidence limits (95% CL). Isobolographic calcula- after acetic acid administration. Antinociceptive activity was tions were performed with the program Pharm Tools Pro (ver- expressed as percent inhibition of the usual number of writhes sion 1.27, The McCary Group Inc.), based on Tallarida (2000). observed in control animals in this study. The statistical difference between theoretical and experimental 24 H.F. Miranda et al. / Pain 121 (2006) 22–28 values was assessed by Student’s t test for independent means Table 1 and P values less than 0.05 (P < 0.05) were considered ED50 values and 95% CL for the antinociceptive effect of NSAIDs in significant. the writhing test of mice Drugs ED50 CL ED50 CL 3. Results mg/kg i.p. mg/kg p.o. Ibuprofen 0.8 0.12–6.1 3.1. Antinociception induced by paracetamol and NSAIDs Parecoxib 1.6 1.0–2.6 Meloxicam 6.5 4.9–8.4 Nimesulide 7.6 5.8–9.8 The i.p.