J Pharmacol Sci 118, 299 – 302 (2012) Journal of Pharmacological Sciences © The Japanese Pharmacological Society Short Communication Synergistic Interaction Between Fentanyl and a Tramadol:Paracetamol Combination on the Inhibition of Nociception in Mice

Víctor Fernández-Dueñas1,*, Raquel Poveda1, Sílvia Sánchez1, and Francisco Ciruela1 1Unit of Pharmacology, Department of Pathology and Experimental Therapeutics, Faculty of Medicine, The University of Barcelona, L’Hospitalet de Llobregat, 08907 Barcelona, Spain

Received September 1, 2011; Accepted December 25, 2011

Abstract. Multimodal analgesic approaches to manage acute and chronic pain are commonly used in humans. Here, we attempted to characterize a synergistic interaction between fentanyl, tramadol, and paracetamol on the inhibition of nociception in a model of visceral pain in mice. The three-drug combined treatment displayed a potent synergistic antinociceptive effect, together with a significant reduction of gastrointestinal transit inhibition. Furthermore, selective μ- and κ-opioid receptor antagonists reversed these synergistic antinociceptive effects, thus suggesting a pivotal role of the opioid system. Overall, this study presents accurate pre-clinical data that might be use- ful to improve the clinical management of opioid-mediated analgesia.

Keywords: opioid multimodal analgesia, drug–drug interaction, synergy

In clinics, the use of drug–drug combinations in order would be enhanced together with a FEN-dosage reduc- to manage acute and chronic pain is well established. On tion and the consequent diminution of opioid-related the one hand, analgesics are staggered according to pain adverse effects (i.e., constipation). In addition, we evalu- severity: mild, moderate, and severe pain, and thus pa- ated the role of opioid receptors in such drug–drug inter- tients should progress through this analgesic ladder de- action, since both TRM and PRC possess diverse and pending on the pain relief obtained. On the other hand, complex mechanisms of action, in which the modulation analgesic adjuvants should also be added in order to of the opioid system would be included (4 – 6). reach analgesia using lower doses of the drugs (i.e., First of all, we analyzed the antinociceptive effects of opioids) used, thus reducing the incidence of adverse the individual drugs. Briefly, male Swiss CD1 mice side-effects. However, although there is precise catego- (20 – 25 g) housed under controlled standard conditions rization of analgesics within drug–drug combinations, (12-h dark/light cycle, 22°C temperature and 66% hu- these multimodal analgesic approaches are most of the midity) were injected with one or a mixture of the fol- times based on empirical instead of accurate experimen- lowing drugs: FEN (KernPharma, Barcelona, Spain); tal data. In such a way, tramadol (TRM) and paracetamol TRM (Grünenthal, Madrid, Spain); PRC (Bristol-Myers (PRC) have been revealed as analgesic drugs that would Squibb, Madrid, Spain); naloxone (Sigma-Aldrich, synergically interact with opioid drugs (1, 2). Further- St. Louis, MO, USA); naltrindole and nor-binaltorphimine more, the combination of TRM and PRC is also a com- (nor-BNI) (Tocris Bioscience, Bristol, UK) subcutane- mon multimodal analgesic strategy that has been shown ously 30 min before testing. Then, mice were injected to be effective and generally well tolerated in patients intraperitoneally with an acetic acid solution (0.6% v/v, with moderate to severe pain (3). Accordingly, in the 10 ml/kg) and placed in a clear plexiglass cylinder to present study we attempted to characterize the combina- observe after 5 min the number of writhes (a single writhe tion of the major opioid fentanyl (FEN) with a is defined as a contraction of the abdominal wall together tramadol:paracetamol (TRM:PRC) mixture, in order to with the twisting of the trunk and hind limbs extension) examine whether the analgesic effects of the mixture in a 5-min period (6). The Institutional Committee on Animal Use and Care, in accordance with the Interna- *Corresponding author. [email protected] tional Association for the Study of Pain guidelines on Published online in J-STAGE on February 3, 2012 (in advance) ethical standards for investigation in animals, approved doi: 10.1254/jphs.11161SC the protocol. As previously described, FEN and PRC

299 300 V Fernández-Dueñas et al induced a dose-dependent inhibition of writhes with analgesic drugs]. Furthermore, the selective κ-opioid similar efficacy (100%) and the following ED50’s: receptor antagonist nor-BNI partially blocked while the FEN = 0.021 ± 0.003, PRC = 109.3 ± 2.5 mg/kg (6). δ-opioid receptor antagonist naltrindole did not affect the Similarly, TRM also dose-dependently inhibited acetic combined treatment–mediated antinociception (Fig. 2A). acid–induced nociception (TRM = 3.18 ± 0.27 mg/kg). Finally, we also evaluated the effects of the combined

Once the individual ED50 values were obtained, TRM treatment on gastrointestinal transit (GIT) inhibition, a and PRC were combined, based on previous studies (7, common side effect of opioid therapy. GIT inhibition 8), in a fixed ratio (1:8) combination (TRM:PRC). The was measured by orally-administrating to 18-h fasted interaction between the different drugs was analyzed animals a charcoal meal (0.25 ml of a suspension of 10% using the previously described procedure (6) based on vegetable charcoal in 5% gum acacia, Sigma-Aldrich) the method developed by Tallarida et al. (9). Thus, the and calculating the percent of the distance traveled by the mixture induced dose-dependent antinociceptive effects, charcoal relative to the total length of the intestine (11). with similar efficacy (100%), and the dose–response Interestingly, when comparing the effects on GIT inhibi- curve was shifted to the left with respect to the theoretical tion of the mixture TRM:PRC plus FEN with that of FEN additive dose–response curve (P < 0.05, Student’s t-test). alone, the dose–response curve of the former was parallel Synergism was also assessed by means of isobolographic and shifted rightward (P < 0.05, Student’s t-test) (Fig. analysis (Fig. 1A), obtaining an interaction index of 2B), thus indicating that the combined treatment dimin- 0.40 ± 0.08. Next, the double combination TRM:PRC ished FEN-mediated GIT inhibition. was treated as a single drug and mixed with FEN in a Overall, our results show that a synergistic interaction fixed ratio (1:1) combination based on ED50 fractions. occurred between the three drugs in the relief of acetic Figure 1B shows the isobologram when adding FEN to acid–mediated nociception in mice and that this multi- the mixture TRM:PRC (1:8). The interaction index modal approach enabled a decrease in FEN dosage and (0.37 ± 0.06) was significantly lower than 1 (P < 0.05, reduced the appearance of adverse effects, namely GIT Student’s t-test), thus clearly demonstrating that these inhibition. First, after testing the antinociceptive effects drugs displayed a potent synergistic effect on the inhibi- of the individual drugs, we evaluated the effects of the tion of acetic acid–mediated nociception. Thereafter, we combination of TRM plus PRC. This multimodal ap- evaluated the effects of selective opioid antagonists on proach has been widely validated in clinics, for instance, the antinociceptive effect of the triple combination. Inter- in the treatment of postsurgical-associated pain (3), while estingly, this antinociceptive effect was completely an- it has not been fully studied in animal models. Interest- tagonized when treating with naloxone [both at non-se- ingly, our results are in accordance with that obtained lective (1 mg/kg) and μ-opioid selective (0.1 mg/kg) recently in a streptozotocin-induced diabetic rat model, doses (10), s.c. administration immediately before the in which a TRM:PRC mixture induced supra-additive

A B

4 0.025

0.02

0.015 2 0.01 Fentanyl (mg/kg) Tramadol (mg/kg) 50 50 ED

ED 0.005

0 0 0 20406080100120 0 50 100 150

ED50 Tramadol-Paracetamol (mg/kg) ED50 Paracetamol (mg/kg)

Fig. 1. Isobolographic representation of the antinociceptive effects of the combination of tramadol plus paracetamol (A) and a

mixture of tramadol:paracetamol (TRM:PRC) in a fixed 1:8 ratio plus fentanyl (FEN) (B), at the ED50 level of effect, in the

writhing test. The filled square in the line joining the x- and y-axes corresponds to the theoretical ED50 with 95% CL; the open

square below the isobole corresponds to the experimental ED50 with 95% CL. There were significant differences between the experimental and the theoretical values (P < 0.05, Student’s t-test). Multimodal Analgesia in Mice 301

A B FEN TRM:PRC + FEN FEN TRM:PRC + FEN 100 100

80 80

60 60

40 * 40 % inhibition

* % GIT inhibition 20 * 20

0 0 0.001 0.01 0.1 1

saline nor-BNI Fentanyl (mg/kg) naloxone naltrindole

Fig. 2. Reversion of the antinociceptive effects, and GIT inhibition, of the triple combination. A) Effects of opioid receptor an- tagonists: naloxone (0.1 mg/kg), naltrindole (3 mg/kg), and nor-binaltorphimine (nor-BNI) (10 mg/kg) on the inhibition of acetic

acid–mediated nociception (% inhibition), assessed by the determined effective dose (ED80) of the individual drugs (FEN = 0.037 mg/kg) and of a mixture containing the three drugs (TRM:PRC plus FEN; TRM = 0.25 mg/kg, PRC = 56.74 mg/kg, FEN = 0.011 mg/kg). In control animals, saline was injected instead of the antagonist. Each column represents mean values of at least eight mice, and vertical bars indicate S.E.M. * indicates statistically significant differences (P < 0.05, Student’s t-test) between saline and antagonist-treated mice for each treatment (FEN, TRM:PRC + FEN). B) Dose–response regression lines for the administra- tion of fentanyl (FEN, squares) and the combination of a fixed (1:8) tramadol:paracetamol dose plus fentanyl (TRM:PRC + FEN, triangles; TRM = 0.14 mg/kg, PRC = 31.26 mg/kg, FEN = 0.01 – 0.1 mg/kg) on the inhibition of gastrointestinal transit (% GIT inhibition). Each point represents mean values of at least eight mice, and vertical bars indicate S.E.M. anti-hyperalgesic effects (12), while other authors did not of action of such drug–drug interaction, our results point find a synergic effect of the TRM:PRC mixture in rats out the opioid system as one of the responsible systems. with neuropathic spinal cord injury pain (13). Needless Interestingly, naloxone completely reversed the antinoci- to say, it would seem likely that the controversies could ceptive effects of the three-drug combined treatment, be explained either by the nociceptive stimuli used or by while the selective δ-opioid receptor antagonist naltrin- the proportion of the drugs in the mixture. Accordingly, dole did not affect them and the selective κ-opioid- the rationale behind the use of the TRM:PRC (1:8) pro- receptor antagonist nor-BNI partially blocked these ef- portion was based on two evidences. First, it was previ- fects. As previously described, PRC as well as the ously shown that only ratios over 1:5.7 were expected to atypical opioid agonist TRM have been suggested to in- be supra-additive in relieving visceral-induced nocicep- duce their antinociceptive effects, along with other tion in mice (7); and second, ratios of approximately 1:8 mechanisms, via opioid receptors (4, 5). It would then have been developed by pharmaceutical companies for seem likely that the antinociceptive effects of the distinct combination therapy in clinics (8). drugs may converge at opioid receptors, either by a direct Subsequently, our aim consisted of performing a activation of the receptors or sensitization of their intra- multimodal analgesic paradigm, in which the three drugs cellular signaling pathways or by an increase in the re- were combined. It is noteworthy that we recently demon- lease of endogenous opioid peptides; accordingly, opioid strated that FEN and PRC interact in a synergic manner receptors (mainly the μ- but also the κ-subtype) would on the inhibition of acetic acid–induced nociception in play a central role integrating the antinociceptive effects mice (6), and similarly FEN and TRM have been shown of the present drug–drug combination. Interestingly, the to induce synergic antinociceptive effects in the writhing role of opioid receptors on the effects of the three-drug test (14), validating the clinical strategy of using opioid– combined treatment was also observed when examining opioid combinations in the management of pain, usually GIT inhibition. Thus, the prominent function of opioid in patients in which opioid tolerance appears (2). On the receptors was further demonstrated by the parallelism of basis of these results, we expected supra-additive anti- the obtained dose–response curves that suggested the nociceptive effects when combining the three drugs, and same mechanism of action in both experimental condi- effectively FEN:TRM:PRC displayed a potent synergis- tions (14). On the other hand, while an anti-transit effect tic interaction on the inhibition of acetic acid–mediated of PRC has not been reported, it is well-described that nociception. On the other hand, regarding the mechanism not only FEN but also TRM, as an atypical opioid ago- 302 V Fernández-Dueñas et al nist, induce constipation (15). Therefore, it has to be also to the mechanism of action of tramadol, an “atypical” opioid an- considered that the diminution of the anti-transit effect algesic. J Pharmacol Exp Ther. 1992;260:275–285. would be mostly mediated by the reduction of the opioid 5 Raffa RB, Walker EA, Sterious SN. Opioid receptors and acet- drugs in the mixture, which in fact was one of the aims aminophen (paracetamol). Eur J Pharmacol. 2004;503:209–210. 6 Fernandez-Duenas V, Poveda R, Fernandez A, Sanchez S, Planas of the present drug–drug combination, reducing undesir- E, Ciruela F. Fentanyl-trazodone-paracetamol triple drug combi- able but increasing antinociceptive effects. nation: multimodal analgesia in a mouse model of visceral pain. In conclusion, our study shows that the combination of Pharmacol Biochem Behav. 2011;98:331–336. FEN, TRM, and PRC leads to synergistic antinociceptive 7 Tallarida RJ, Raffa RB. Testing for synergism over a range of effects and that μ- and κ-opioid receptors play a central fixed ratio drug combinations: replacing the isobologram. Life role in such drug–drug interaction. Furthermore, this Sci. 1996;58:PL23–PL28. multimodal analgesic approach permits reducing possible 8 Lee EY, Lee EB, Park BJ, Lee CK, Yoo B, Lim MK, et al. Trama- dol 37.5-mg/acetaminophen 325-mg combination tablets added undesired secondary opioid-associated effects. We have to regular therapy for rheumatoid arthritis pain: a 1-week, ran- therefore characterized a common multimodal analgesic domized, double-blind, placebo-controlled trial. Clin Ther. 2006; strategy used in clinics, validating this approach for the 28:2052–2060. management of pain in which opioid drugs are used. 9 Tallarida RJ. Drug synergism: its detection and applications. J Pharmacol Exp Ther. 2001;298:865–872. Acknowledgments 10 Magnam J, Paterson SJ, Tavani A, Kosterlitz HW. The binding spectrum of narcotic analgesic drugs with different agonist and The authors belong to the “Neuropharmacology and Pain” accred- antagonist properties. Naunyn Schmiedebergs Arch Pharmacol. ited research group (Generalitat de Catalunya, 2009 SGR 232). This 1982;319:197–205. work was supported by grants SAF2008-01462 and Consolider- 11 Puig MM, Pol O. 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