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Isobolographic analysis of the dual-site synergism in the antinociceptive response of tramadol in the formalin test in rats
Life Sciences 79 (2006) 2275 – 2282 www.elsevier.com/locate/lifescie
Isobolographic analysis of the dual-site synergism in the antinociceptive response of tramadol in the formalin test in rats Amaury J. Pozos-Guillén a , Patricia Aguirre-Bañuelos b , Abraham Arellano-Guerrero b , Gilberto Castañeda-Hernández c , Carlos Hoyo-Vadillo c , José Pérez-Urizar b,⁎ b
a Facultad de Estomatología, Universidad Autónoma de San Luís Potosí, San Luís Potosí, México Facultad de Ciencias Químicas, Universidad Autónoma de San Luís Potosí, San Luís Potosí, México c Sección Externa de Farmacología, CINVESTAV, México D.F., México
Received 21 September 2005; accepted 27 July 2006
Abstract Tramadol is an atypical opioid with a complex mechanism of action including a synergistic interaction between the parent drug and an active metabolite. The local action of the parent drug is poorly documented. This study was designed to evaluate the site–site interaction of the antinociception produced by tramadol given by two different routes. The effects of individual and fixed-ratio combinations of intraplantar (i.pl.) and intraperitoneal (i.p.) tramadol were evaluated using the formalin test in rats. Isobolographic analysis was employed to identify the synergy produced by combinations. In both first and second phases of the formalin test, tramadol was active not only by the systemic (ED50 10.2 ± 2.1 and 7.1 ± 0.5 mg/kg i.p.) but also by the local route (ED50 171.0 ± 44.8 and 134.6 μg/paw i.pl.). The isobolographic analysis revealed a “selfsynergism” in the antinociceptive effect between the two routes of administration, as the experimental ED50 (211.1 ± 13.6 and 45.9 ± 3.9 “dose units” phase 1 and 2, respectively) of the combination was significantly lower than the theoretical ED50 (422.2 ± 50.5 and 138.5 ± 9.2 “dose units”). The mechanism underlying this self-synergism appears to be partially opioid since systemic but not local naloxone reversed the potentiation. The observed dual-site interaction in the antinociceptive action of tramadol provides insights for alternatives in the management of pain. © 2006 Elsevier Inc. All rights reserved. Keywords: Synergistic interaction; Tramadol; Antinociception; Self-synergism
Introduction Tramadol hydrochloride, (1RS, 2RS)-2-((dimethyl amino)methyl)-1-(3-methoxyphenyl)-cyclohexanol hydrochloride, is clinically effective in the treatment of moderate to severe pain with a relatively low addiction incidence. In acute therapeutic use, tramadol produces analgesia against multiple pain conditions, such as postsurgical pain, obstetric pain, terminal cancer pain and pain of coronary origin, and it has been used as adjuvant therapy in anesthesia (Scott and Perry, 2000). It acts at opioid receptors and also appears to modify the transmission of pain impulses by ⁎ Corresponding author. Laboratorio de Farmacología y Fisiología, Facultad de Ciencias Químicas, Universidad Autónoma de San Luís Potosí Av. Dr. Nava # 6. Zona Universitaria, San Luís Potosí, México. 78200. Tel.: +52 444 8262440x520; fax: +52 444 8262372. E-mail address: [email protected] (J. Pérez-Urizar). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.07.029
inhibition of monoamine reuptake. Indeed, Bamigbade et al. (1997) showed that (±)-tramadol and its (+)-enantiomer not only blocked the dorsal raphe nucleus 5-HT uptake but also increased the 5-HT efflux. This activity, at clinically relevant concentrations, may help to explain the antinociceptive efficacy of tramadol despite weak μ-opioid receptor affinity and adds to the evidence that tramadol exerts actions on central monoaminergic systems that may contribute to its analgesic effect. Raffa et al. (1993) demonstrated that racemic tramadol was significantly more potent than the theoretical additive effect of the enantiomers in different experimental models of nociception (antinociceptive synergy). While Valle et al. (2000) elegantly described a pk/pd model for the generation and antinociceptive interaction between the (+)- and the (−)-O-desmethyltramadol (M1) metabolite suggesting that the systemic action of tramadol results from the complementary interaction between the enantiomers of the parent drug and its M1 metabolite.
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There are clinically important side effects related to opioid pain therapy. Limiting drug exposure to the periphery would reduce central side effects such as sedation, respiratory depression, and nausea (Kolesnikov et al., 2000). In that regard, a number of studies have demonstrated that topically applied morphine provides effective analgesia without adverse effects, including tolerance, in patients and rodents under painful inflammatory conditions (Watterson et al., 2004; Junien and Wettstein, 1992). However, in the case of tramadol, few studies have provided evidence that this drug exerts a local effect (Tsai et al., 2001; Pang et al., 1998; Altunkaya et al., 2003). In fact, the peripheral action of tramadol has been assessed only as an anesthetic adjuvant but not as an analgesic agent (Kapral et al., 1999). Indeed, Tsai et al. (2001) demonstrated the blockage of neural conduction by the direct application of tramadol on the sciatic nerves of rats. Interestingly, the local anesthetic effect of tramadol was not reversed by naloxone suggesting that an underlying mechanism other than opioid activation is present. Drug combination is another approach used to obtain effective analgesia while decreasing the incidence and severity of side effects. Simultaneous administration of two drugs can produce an additive effect, though more relevant is that the observed effect can be of greater/lesser magnitude resulting in synergism or antagonism. Indeed, in a previous review of different settings of clinical acute pain Kehlet et al. (1999) defined the concept of balanced or multimodal analgesia what implies a combination of different classes of analgesics as well as the use of different sites of administration of analgesics in order to improve pain relief. In that sense, tramadol has been successfully combined with several selective and unselective cyclooxygenase inhibitors as well as with other adjuvants to alleviate experimental and clinical pain (Satyanarayana et al., 2004; Lopez-Munoz et al., 2004; Schnitzer, 2003; Chen et al., 2002). The concept of balanced analgesia has been extended to describe the synergism observed after the administration of even a single agent by different routes (also called “self-synergism”) (Tallarida, 2001). For instance, by using isobolographic analysis, Raffa et al. (2000) showed that the spinal plus supraspinal administration of acetaminophen produced antinociception greater than that expected by simply the sum of individual doses. The authors proposed that site–site interaction was produced by the acetaminophen-stimulated release of an opioid-like substance supraspinally but not spinally. Despite the lower incidence and severity of opioid-like side effects associated with tramadol compared with other, stronger opioid agents, disturbances such as the appearance of serotonergic syndrome should be considered before prescribing the analgesic (Scott and Perry, 2000). Serotonergic syndrome is characterized by tachycardia, confusion, psychosis, sundowning, agitation, diaphoresis, and tremor. It has been proposed that all drugs that directly or indirectly increase central serotonin neurotransmission at postsynaptic 5-HT(1A) and 5-HT(2A) receptors can produce the serotonergic syndrome (Houlihan, 2004; Rojas-Corrales et al., 2005). Thus, since the (+)-enantiomer of tramadol inhibits serotonin uptake and due to the growing use of tramadol in different states of acute and chronic pain, it would be valuable to develop strategies that permit the safer use of this atypical opioid agent.
Therefore, the present study was designed, first, to determine the local antinociceptive effect of tramadol and second, to evaluate the possible site–site synergism in the antinociception produced by tramadol given to rats simultaneously by intraplantar (local) and intraperitoneal (systemic) routes in the formalin test. Material and methods Animals Male Wistar rats aged 6–7 weeks (weight range, 150–200 g) from our own breeding facilities were used in this study. Rats were housed at 22 °C with a 12-h light/dark cycle and had free access to food and tap water up to the time of the experiment. Each rat 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 Investigation of Experimental Pain in Animals” (Zimmermann, 1983). Drugs Tramadol hydrochloride was kindly donated by Dr. Iñaki F. Troconiz (University of Navarra, Spain). Naloxone was obtained from Sigma–Aldrich Co. (St. Louis, MO). To evaluate the local effect of tramadol and naloxone, both agents were dissolved in saline and injected by the intraplantar route (i.pl.). The total volume of injection to the rat paw with formalin alone or combined with drugs was 50 μl. For intraperitoneal (i.p.) administration, drugs were also dissolved in saline and given at a volume ratio of 1 ml/kg 15 min before the formalin injection. Control groups of rats received only saline solution. Measurement of nociceptive response Antinociception was assessed by the formalin test. Rats were placed in clear plastic chambers with a mirror placed at a 45° angle to allow an unobstructed view of the paws. The rats were injected into the dorsal surface of the hind paw with 50 μl of dilute formalin (5%) alone or mixed with the appropriated concentrations of agents using a 30-gauge needle. The rats were observed Table 1 Dosing scheme of tramadol in the dual-site interaction study when given by both routes Combination
(Local ED50) + (systemic ED50) (Local ED50/2) + (systemic ED50/2) (Local ED50/4) + (systemic ED50/4) (Local ED50/8) + (systemic ED50/8) a
See text for details.
Route combination (“dose units”)a
Composition of the route combination Local dose (μg/paw)
Systemic dose (mg/kg)
141.8
134.6
7.2
70.8
67.3
3.6
35.4
33.6
1.8
17.7
16.8
0.9
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data were quantified as the percent of reduction in flinches relative to the control total in each phase (first, 0–15 min and second 15– 60 min) of the formalin test. Percent of antinociception was calculated according to the following equation: %Antinociception ¼ ððControl−DrugÞ=ControlÞ 100 Where control and drug refer to the number of flinches elicited by formalin plus saline and formalin plus drug, respectively. Experimental design Dose–response curves of tramadol-mediated antinociception following systemic (3, 10, 50 mg/kg i.p.) and local (50, 100, 250, 400 μg/paw i.pl.) administration were assessed and the respective ED50 ± standard error values were determined (Tallarida, 2000). To assess if the antinociception following intraplantar tramadol was due to local action, formalin was administered in one paw and the tested drug in the contralateral paw. Moreover to evaluate if the local effect of tramadol underlies an opioid mechanism of action, naloxone (100 μg/paw) was simultaneously injected by the intraplantar pathway, and results were compared to those obtained with an effective peripheral dose of morphine (5 μg/paw). Local doses of naloxone and morphine were chosen from pilot experiments or from previous reports (Mixcoatl-Zecuatl et al., 2000). Since no significant differences were seen between the ED50's for the two phases of the test, subsequent experiments were based upon the ED50 data from the second phase of the formalin test. Thus a dose–response curve was constructed from the concurrent delivery of the two routes of administration in a constant dose ratio of ED50's (local pathway ED50 = 134.6 μg/paw + systemic pathway ED50 = 7.2 mg/kg), and subsequent dilutions thereafter (local ED50/2 + systemic ED50/2, local ED50/4 + systemic ED50/4 and local ED50/8 + systemic ED50/8), as shown in Table 1. In order
Fig. 1. Time-course of the antinociceptive effect of (A) local (peripheral) administration of tramadol 3 (○), 10 (∇) and 50 (▾) mg/kg i.p or saline (●); (B) systemic administration of tramadol 50 (○), 100 (∇), 250 (▾) and 400 (◼) μg/ paw i.pl. or saline (●); and (C) local–systemic combination of tramadol 17.7 (○), 35.4 (∇), 70.8 (▾) and 141.8 (◼) “dose units” (see text for details) or saline (●). Data represent the mean ± S.E.M. of 6 rats.
for nociceptive behavior immediately after formalin injection. Nociceptive behavior was quantified as the number of flinches of the injected paw 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
Fig. 2. Evidence of the significant antinociceptive effect of ipsilateral (IL) but not contralateral (CL) locally administered tramadol (400 μg/paw). This effect is not reversed by naloxone (100 μg/paw) in contrast to an effective dose of morphine (100 μg/paw) in the second phase of the formalin test. Results are presented as the mean of 6 rats (± S.E.M.). ⁎ Denotes differences from the formalin untreated group (p b 0.05).
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(Tallarida, 2000). Briefly, the ED50 values of each route were plotted on the x- and y-axes, respectively. The theoretically additive dose combination was calculated. From the variance of the total dose, individual variances for each route in the combination were obtained. To further describe the magnitude of the interaction, the interaction index (γ) (Tallarida, 2002) was calculated as: ((ED50 dose in combination for route 1))/(ED50 value for route 1 given alone)) + ((ED50 dose in combination for route 2)/(ED50 value for route 2 given alone)). A value of 1 indicates an additive interaction whereas a value b 1 indicates a synergistic interaction and a value N1 implies an antagonistic interaction. Finally to evaluate the opioid component underlying “selfsynergism”, local (100 μg/paw i.pl.) and systemic (1 mg/kg i.p.) naloxone was administered simultaneously with the ED50 of the route combination. Data analysis and statistics Results are presented as the mean ± S.E.M. for 6–8 animals per group. Statistical significance between the theoretical additive point and the experimentally-derived value was evaluated by using Student's t-test. p b 0.05 was considered to indicate a significant synergistic interaction between the two routes of administration (Tallarida, 2000, 2001). Data from the experiments testing the role of the opioid pathway in the mechanism of self-synergism were analyzed using the one way ANOVA followed by the Tukey test. Results Local and systemic antinociceptive effect of tramadol
Fig. 3. Comparative dose–effect curves for the intraperitoneal (A) and intraplantar (B) administration of (±)-tramadol evaluated in the first (open circles) and the second phase (filled circles) of the formalin test. Data are shown as the mean (n = 6) ± S.E.M.
to correctly handle the data from such combinations, a dummy variable of dosing was introduced, namely “dose units” since the constituent dose units, μg/paw (local) and mg/kg (systemic) cannot be mixed, so that “dose units” corresponds to the arithmetical sum of an intraplantar (μg/paw) plus an intraperitoneal (mg/kg) dose combination of tramadol. In order to evaluate the nature of any site–site antinociceptive interaction between the two routes of administration, isobolograms were constructed using doses producing 50% maximum possible effect (ED50) obtained when tramadol was administered for each individual route or combined as previously described
Subcutaneous injection of formalin into the hind paw produced a biphasic pattern of flinching behavior. The first phase started immediately after administration of formalin and then diminished gradually in approximately 10 min. The second phase started at 15 min and lasted until 1 h. Fig. 1(A–C) shows the time-courses of flinching behavior in control and treated rats with increasing doses of tramadol given by local, systemic and both routes. Local ipsilateral, but not contralateral administration of single or dualsite tramadol produced a significant (p b 0.05) reduction in the flinching behavior otherwise observed after formalin injection,
Table 2 Effect of tramadol administered by local (intraplantar) and systemic (intraperitoneal) routes alone and in combination in both phases of the formalin test
Local (μg/paw) Systemic (mg/kg) Theoretical combination (“dose units”) Experimental combination (“dose units”) Interaction index
First phase ED50
Second phase ED50
171.0 ± 44.7 10.2 ± 2.1 422.2 ± 50.5 211.1 ± 13.6⁎ 0.50 ± 0.06
134.6 ± 25.1 7.1 ± 0.5 138.5 ± 9.2 45.9 ± 3.9⁎ 0.33 ± 0.03
ED50: effective dose resulting in a 50% reduction in the nociceptive response. Data are the mean ± S.E.M. ⁎Significantly different from the theoretical combination data (p b 0.05), by the Student's t-test.
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Tramadol was able to produce a dose-dependent suppression of the flinching response in both phases of the formalin test not only after the intraperitoneal but also following the intraplantar dosing, although the antinociception was superior following the systemic administration (Fig. 3A–B). In addition, no differences in the analgesic potency (ED50) of tramadol between the first and second phases of the test were noticed following administration by either route (Table 2). Therefore, the experimental design of the isobolographic analysis was based on the ED50's for the second phase. In order to detect spontaneous central side effects, an assessment of behavioral neurotoxicity was performed as described by Yamamoto and Yaksh (1992). No abnormality of placing, stepping, or righting reflexes was exhibited in the rats before or after treatments in either group: control or treated by any of the studied routes of administration (data not shown). Isobolographic analysis of the dual-site synergism in the antinociceptive effect of tramadol
Fig. 4. Isobolograms of the ED50 values for tramadol administered concurrently by the systemic (intraperitoneal) and local (intraplantar) pathways in the first (A) and the second phase of the formalin test. Horizontal and vertical bars indicate S.E.M. The oblique line between the x- and y-axis is the theoretical additive line. The point in the middle of this line, indicated by “T”, is the theoretical doseadditive point calculated from the separate ED50 values. The point indicated by “E” is the experimentally derived ED50 value observed with the combination. In all cases the experimental point is situated below the additive line, indicating a significant synergism (p b 0.05).
suggesting that the response was actually elicited by local mechanisms (Fig. 2). Moreover this effect was not reversed by naloxone (100 μg/paw) in contrast to an effective dose of morphine (100 μg/paw) in the second phase of the formalin test.
Tramadol was also able to produce a dose-dependent suppression of the flinching response in both phases of the formalin test following the systemic–local route combination at a fixed ratio (Fig. 3C). The isobolographic analysis of the dual-site interaction in the antinociception produced by tramadol given by the two different routes is shown on Fig. 4. In both phases of the formalin test the experimental point lies far below the additive line indicating a significant synergism (p b 0.05). In addition, the experimental ED50 values for the routes in combination were significantly lower than the theoretical ED50 by factors of 2 and 3 in phase 1 and 2 of the test, respectively (Table 2). Moreover, the selfsynergistic interaction between the two routes of administration was confirmed from the estimation of the interaction index, for which the values of 0.5 and 0.3 in phases 1 and 2 of the formalin test were far less than 1 (Table 2).
Fig. 5. The dual-site synergized tramadol antinociception is reversed by systemic (1 mg/kg i.p.; NLXS) or combined local + systemic naloxone (100 μg/paw i.pl. + 1 mg/kg i.p.), but not by local naloxone (100 μg/paw i.pl.; NLXS) in the second phase of the formalin test (B). Neither local (100 μg/paw i.pl.) nor systemic (1 mg/kg i.p.) naloxone by itself modified the natural nociceptive response to the intraplantar injection of formalin (open bars). Results are presented as the mean of 6 rats (±S.E.M.). ⁎p b 0.05 vs formalin untreated group (saline); ⁎⁎p b 0.05 vs the TMDL + TMDS (dual-site tramadol combination) group.
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Role of the opioid pathway in the dual-site synergism of tramadol antinociception In order to evaluate whether peripheral and/or central opioid mechanisms were involved in the dual-site synergism, local (100 μg/paw i.pl.) and systemic (1 mg/kg i.p.) naloxone was administered alone or jointly with the experimental ED50 of the combination (211 and 46 “dose units” for phases 1 and 2 of the formalin test, respectively). Neither local nor systemic naloxone by itself modified the formalin-evoked nociceptive response (Fig. 5A). Local naloxone (100 μg/paw i.pl.) was unable to reverse the increased antinociceptive effect of the combination in the experimental model. In contrast systemic naloxone (1 mg/ kg i.p.) alone or combined with local naloxone partially blocked the increased antinociception of tramadol given by two routes (Fig. 5B) in the second phase of the formalin test. Discussion Management of acute and chronic pain is important, not only for patient well-being but also to prevent long-term complications and morbidity (APSQCC, 1995). Although the analgesic efficacy of tramadol has been shown, side effects, particularly opioidrelated, remain a concern (Silvasti et al., 1999). In order to overcome this, new therapeutic strategies, such as balanced analgesia, have been developed (Kehlet et al., 1999). In the present study we found that the adaptation of the balanced analgesia concept to the combination of multiple routes of administration of a single agent, namely local and systemic tramadol, promises to maintain a good antinociceptive effect with reduced central and systemic exposure to the opioid. There are many descriptions of tramadol analgesia after its systemic administration (Lee et al., 1993; Scott and Perry, 2000), but little evidence regarding the peripheral action of tramadol is available. The latter is important because by limiting the exposure of a drug to the periphery, opioid-mediated central side effects can be markedly reduced. In that sense, although previous findings suggested a role for tramadol as a potential local anesthetic agent (Pang et al., 1998; Altunkaya et al., 2003), to our knowledge this is the first report showing that tramadol is able to produce local antinociception in an experimental model of acute and tonic pain. In this study we found that intraplantar tramadol was able to dose-dependently diminish the nociceptive behavior of flinching in both phases of the formalin test in rats by an unclear mechanism of action. One possible explanation is that tramadol by itself interacts with opioid receptors in the primary afferent. However, several lines of evidence support alternative mechanisms. Indeed, previous findings have shown that other opioid agents produce analgesic effects due to an increased density of opioid receptors on peripheral sensory nerves during painful inflammatory processes (Junien and Wettstein, 1992; Watterson et al., 2004). However, tramadol binding affinity for opioid receptors appears to be too low to account for the antinociceptive effects through this system (Lee et al., 1993). Moreover, it is known that the (+)enantiomer of the active metabolite of tramadol is about 200-fold more potent in activating the μ-opioid receptor than the parent
drug, but no tramadol-metabolizing enzymes are present in the inflamed tissue (Gillen et al., 2000). In addition, local naloxone was unable to block the increased antinociceptive response produced by local or the local–systemic tramadol combination in contrast to that observed with an effective dose of local morphine. Therefore, a mechanism other than opioid peripheral pathway activation must be responsible for the local antinociceptive action of tramadol. In that regard, it was postulated that tramadol has a local anesthetic effect similar to that of lidocaine following intradermal injection (Pang et al., 1998). On the other hand, tramadol appears to have anti-inflammatory properties independent of the arachidonic acid pathway (Bianchi et al., 1999). Once confirmed the noticeable local antinociceptive effect of tramadol, the hypothesis that the concurrent administration of the drug by local and systemic pathways produces an increased antinociceptive response was tested. The experimental design was based upon isobolographic analysis using a fixed ratio (19:1 local–systemic dosing) as this methodology is well-recognized as the standard to evaluate drug combinations (Tallarida, 2000). Our results demonstrated a marked self-synergism in the antinociceptive effect between the two routes of administration, i.e. the observed ED50 of the combination was 2 and 3 times (phase 1 and 2 of the formalin test, respectively), less than the theoretical ED50. These observations imply that systemic exposure to tramadol may be reduced up to 3 times, if combined with local administration (e.g. topical formulation or infiltration in wounds), and still retain its antinociceptive efficacy. We used the formalin test to evaluate the antinociception of the tramadol site–site interaction. The observed biphasic pattern reflects different pathological processes, different sensitivities to centrally and peripherally acting analgesic agents, and perhaps different pain qualities (Abbott et al., 1995). Notwithstanding, we found that the potency (ED50) of tramadol administered by the local or systemic routes was not significantly different between the first and second phases of the formalin test. Therefore, the experimental design of the isobolographic analysis was based on the ED50 for the second phase (Yoon and Yaksh, 1999). However, the evaluation of the opioid contribution to the synergism showed that the mixed local–systemic administration of naloxone was able to completely block the increased analgesic effect in the first phase (data not shown). In contrast, only a partial blocking of the synergism was seen in the second phase of the formalin test. The site–site synergism is a phenomenon that has been demonstrated previously. Indeed, Raffa et al. (2000) demonstrated the dual-site analgesic synergism of acetaminophen following the spinal and supraspinal administration of the drug. The authors proposed that the systemic administration of the drug will lead to the spinal release of an opiod-like compound and would explain the self-synergism of acetaminophen in mice. That is, the finding of synergism may not only identify a potential therapeutic use of a drug combination, but also may illuminate the mechanism of action of a drug mixture and even of a single agent (Tallarida, 2001). In this study, although the actual mechanism underlying the synergism remains unclear, the opioid blockade of the antinociceptive response with systemic, but not local naloxone suggests
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that the opioid pathway is not the only mechanism of the potentiation. Furthermore, it is well-known that tramadol-induced analgesia is not accounted for by a single mechanism of action. In fact, following the systemic administration of tramadol, the parent drug and an active metabolite interact with each other by opioid and monoaminergic pathways to reduce the nociceptive input to the spinal cord (Raffa et al., 1993; Valle et al., 2000; Garrido et al., 2003). Thus, in addition to the previously mentioned local anesthetic and anti-inflammatory properties of local tramadol, other mechanisms may be participating. For instance, it has been recently shown that local antidepressants with monoaminergic actions are able to produce analgesia in experimental models of tonic and neuropathic pain (Sawynok et al., 1999; Esser and Sawynok, 1999). Experiments testing the ability of tramadol to inhibit the reuptake of norepinephrin and serotonin at the periphery (currently underway) might help to elucidate the observed self-synergism. In conclusion, this study illustrated for the first time the local peripheral antinociceptive effect of tramadol in a model of acute pain. Our study is the first to demonstrate self-synergism in antinociception by tramadol when administered by two different routes. The observed site–site interaction provides insights for alternatives in the management of pain. Notwithstanding, in order to clarify the mechanism of tramadol local action, as well as that of synergistic combination, further studies should be conducted. Acknowledgements This study was partially supported by the following grants CONACYT-38940-M, PROMEP-103.5/02/2351 and C0-FAI04-3.4. A.J. Pozos-Guillen and P. Aguirre-Bañuelos are CONACYT fellows. We would like to thank Dr. Paul Kretchmer ([email protected]) at San Francisco Edit for his assistance in editing this manuscript. References Abbott, F.V., Franklin, K.B., Westbrook, R.F., 1995. The formalin test: scoring properties of the first and second phases of the pain response in rats. Pain 60, 91–102. Altunkaya, H., Ozer, Y., Kargi, E., Babuccu, O., 2003. Comparison of local anaesthetic effects of tramadol with prilocaine for minor surgical procedures. British Journal of Anaesthesia 90, 320–322. American Pain Society Quality of Care Committee Quality, 1995. Improvement guidelines for the treatment of acute pain and cancer pain. JAMA 274, 1874–1880. Bamigbade, T.A., Davidson, C., Langford, R.M., Stamford, J.A., 1997. Actions of tramadol, its enantiomers and principal metabolite, O-desmethyltramadol, on serotonin (5-HT) efflux and uptake in the rat dorsal raphe nucleus. British Journal of Anaesthesia 79, 352–356. Bianchi, M., Rossoni, G., Sacerdote, P., Panerai, A.E., 1999. Effects of tramadol on experimental inflammation. Fundamental & Clinical Pharmacology 13, 220–225. Chen, Y., Chan, S.Y., Ho, P.C., 2002. Isobolographic analysis of the analgesic interactions between ketamine and tramadol. Journal of Pharmacy and Pharmacology 54, 623–631. Esser, M.J., Sawynok, J., 1999. Acute amitriptyline in a rat model of neuropathic pain: differential symptom and route effects. Pain 80, 643–653. Garrido, M.J., Sayar, O., Segura, C., Rapado, J., Dios-Vieitez, M.C., Renedo, M.J., Troconiz, I.F., 2003. Pharmacokinetic/pharmacodynamic modeling of the antinociceptive effects of (+)-tramadol in the rat: role of cytochrome P450 2D
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Isobolographic analysis of the dual-site synergism in the antinociceptive response of tramadol in the formalin test in rats Amaury J. Pozos-Guillén a , Patricia Aguirre-Bañuelos b , Abraham Arellano-Guerrero b , Gilberto Castañeda-Hernández c , Carlos Hoyo-Vadillo c , José Pérez-Urizar b,⁎ b
a Facultad de Estomatología, Universidad Autónoma de San Luís Potosí, San Luís Potosí, México Facultad de Ciencias Químicas, Universidad Autónoma de San Luís Potosí, San Luís Potosí, México c Sección Externa de Farmacología, CINVESTAV, México D.F., México
Received 21 September 2005; accepted 27 July 2006
Abstract Tramadol is an atypical opioid with a complex mechanism of action including a synergistic interaction between the parent drug and an active metabolite. The local action of the parent drug is poorly documented. This study was designed to evaluate the site–site interaction of the antinociception produced by tramadol given by two different routes. The effects of individual and fixed-ratio combinations of intraplantar (i.pl.) and intraperitoneal (i.p.) tramadol were evaluated using the formalin test in rats. Isobolographic analysis was employed to identify the synergy produced by combinations. In both first and second phases of the formalin test, tramadol was active not only by the systemic (ED50 10.2 ± 2.1 and 7.1 ± 0.5 mg/kg i.p.) but also by the local route (ED50 171.0 ± 44.8 and 134.6 μg/paw i.pl.). The isobolographic analysis revealed a “selfsynergism” in the antinociceptive effect between the two routes of administration, as the experimental ED50 (211.1 ± 13.6 and 45.9 ± 3.9 “dose units” phase 1 and 2, respectively) of the combination was significantly lower than the theoretical ED50 (422.2 ± 50.5 and 138.5 ± 9.2 “dose units”). The mechanism underlying this self-synergism appears to be partially opioid since systemic but not local naloxone reversed the potentiation. The observed dual-site interaction in the antinociceptive action of tramadol provides insights for alternatives in the management of pain. © 2006 Elsevier Inc. All rights reserved. Keywords: Synergistic interaction; Tramadol; Antinociception; Self-synergism
Introduction Tramadol hydrochloride, (1RS, 2RS)-2-((dimethyl amino)methyl)-1-(3-methoxyphenyl)-cyclohexanol hydrochloride, is clinically effective in the treatment of moderate to severe pain with a relatively low addiction incidence. In acute therapeutic use, tramadol produces analgesia against multiple pain conditions, such as postsurgical pain, obstetric pain, terminal cancer pain and pain of coronary origin, and it has been used as adjuvant therapy in anesthesia (Scott and Perry, 2000). It acts at opioid receptors and also appears to modify the transmission of pain impulses by ⁎ Corresponding author. Laboratorio de Farmacología y Fisiología, Facultad de Ciencias Químicas, Universidad Autónoma de San Luís Potosí Av. Dr. Nava # 6. Zona Universitaria, San Luís Potosí, México. 78200. Tel.: +52 444 8262440x520; fax: +52 444 8262372. E-mail address: [email protected] (J. Pérez-Urizar). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.07.029
inhibition of monoamine reuptake. Indeed, Bamigbade et al. (1997) showed that (±)-tramadol and its (+)-enantiomer not only blocked the dorsal raphe nucleus 5-HT uptake but also increased the 5-HT efflux. This activity, at clinically relevant concentrations, may help to explain the antinociceptive efficacy of tramadol despite weak μ-opioid receptor affinity and adds to the evidence that tramadol exerts actions on central monoaminergic systems that may contribute to its analgesic effect. Raffa et al. (1993) demonstrated that racemic tramadol was significantly more potent than the theoretical additive effect of the enantiomers in different experimental models of nociception (antinociceptive synergy). While Valle et al. (2000) elegantly described a pk/pd model for the generation and antinociceptive interaction between the (+)- and the (−)-O-desmethyltramadol (M1) metabolite suggesting that the systemic action of tramadol results from the complementary interaction between the enantiomers of the parent drug and its M1 metabolite.
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There are clinically important side effects related to opioid pain therapy. Limiting drug exposure to the periphery would reduce central side effects such as sedation, respiratory depression, and nausea (Kolesnikov et al., 2000). In that regard, a number of studies have demonstrated that topically applied morphine provides effective analgesia without adverse effects, including tolerance, in patients and rodents under painful inflammatory conditions (Watterson et al., 2004; Junien and Wettstein, 1992). However, in the case of tramadol, few studies have provided evidence that this drug exerts a local effect (Tsai et al., 2001; Pang et al., 1998; Altunkaya et al., 2003). In fact, the peripheral action of tramadol has been assessed only as an anesthetic adjuvant but not as an analgesic agent (Kapral et al., 1999). Indeed, Tsai et al. (2001) demonstrated the blockage of neural conduction by the direct application of tramadol on the sciatic nerves of rats. Interestingly, the local anesthetic effect of tramadol was not reversed by naloxone suggesting that an underlying mechanism other than opioid activation is present. Drug combination is another approach used to obtain effective analgesia while decreasing the incidence and severity of side effects. Simultaneous administration of two drugs can produce an additive effect, though more relevant is that the observed effect can be of greater/lesser magnitude resulting in synergism or antagonism. Indeed, in a previous review of different settings of clinical acute pain Kehlet et al. (1999) defined the concept of balanced or multimodal analgesia what implies a combination of different classes of analgesics as well as the use of different sites of administration of analgesics in order to improve pain relief. In that sense, tramadol has been successfully combined with several selective and unselective cyclooxygenase inhibitors as well as with other adjuvants to alleviate experimental and clinical pain (Satyanarayana et al., 2004; Lopez-Munoz et al., 2004; Schnitzer, 2003; Chen et al., 2002). The concept of balanced analgesia has been extended to describe the synergism observed after the administration of even a single agent by different routes (also called “self-synergism”) (Tallarida, 2001). For instance, by using isobolographic analysis, Raffa et al. (2000) showed that the spinal plus supraspinal administration of acetaminophen produced antinociception greater than that expected by simply the sum of individual doses. The authors proposed that site–site interaction was produced by the acetaminophen-stimulated release of an opioid-like substance supraspinally but not spinally. Despite the lower incidence and severity of opioid-like side effects associated with tramadol compared with other, stronger opioid agents, disturbances such as the appearance of serotonergic syndrome should be considered before prescribing the analgesic (Scott and Perry, 2000). Serotonergic syndrome is characterized by tachycardia, confusion, psychosis, sundowning, agitation, diaphoresis, and tremor. It has been proposed that all drugs that directly or indirectly increase central serotonin neurotransmission at postsynaptic 5-HT(1A) and 5-HT(2A) receptors can produce the serotonergic syndrome (Houlihan, 2004; Rojas-Corrales et al., 2005). Thus, since the (+)-enantiomer of tramadol inhibits serotonin uptake and due to the growing use of tramadol in different states of acute and chronic pain, it would be valuable to develop strategies that permit the safer use of this atypical opioid agent.
Therefore, the present study was designed, first, to determine the local antinociceptive effect of tramadol and second, to evaluate the possible site–site synergism in the antinociception produced by tramadol given to rats simultaneously by intraplantar (local) and intraperitoneal (systemic) routes in the formalin test. Material and methods Animals Male Wistar rats aged 6–7 weeks (weight range, 150–200 g) from our own breeding facilities were used in this study. Rats were housed at 22 °C with a 12-h light/dark cycle and had free access to food and tap water up to the time of the experiment. Each rat 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 Investigation of Experimental Pain in Animals” (Zimmermann, 1983). Drugs Tramadol hydrochloride was kindly donated by Dr. Iñaki F. Troconiz (University of Navarra, Spain). Naloxone was obtained from Sigma–Aldrich Co. (St. Louis, MO). To evaluate the local effect of tramadol and naloxone, both agents were dissolved in saline and injected by the intraplantar route (i.pl.). The total volume of injection to the rat paw with formalin alone or combined with drugs was 50 μl. For intraperitoneal (i.p.) administration, drugs were also dissolved in saline and given at a volume ratio of 1 ml/kg 15 min before the formalin injection. Control groups of rats received only saline solution. Measurement of nociceptive response Antinociception was assessed by the formalin test. Rats were placed in clear plastic chambers with a mirror placed at a 45° angle to allow an unobstructed view of the paws. The rats were injected into the dorsal surface of the hind paw with 50 μl of dilute formalin (5%) alone or mixed with the appropriated concentrations of agents using a 30-gauge needle. The rats were observed Table 1 Dosing scheme of tramadol in the dual-site interaction study when given by both routes Combination
(Local ED50) + (systemic ED50) (Local ED50/2) + (systemic ED50/2) (Local ED50/4) + (systemic ED50/4) (Local ED50/8) + (systemic ED50/8) a
See text for details.
Route combination (“dose units”)a
Composition of the route combination Local dose (μg/paw)
Systemic dose (mg/kg)
141.8
134.6
7.2
70.8
67.3
3.6
35.4
33.6
1.8
17.7
16.8
0.9
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data were quantified as the percent of reduction in flinches relative to the control total in each phase (first, 0–15 min and second 15– 60 min) of the formalin test. Percent of antinociception was calculated according to the following equation: %Antinociception ¼ ððControl−DrugÞ=ControlÞ 100 Where control and drug refer to the number of flinches elicited by formalin plus saline and formalin plus drug, respectively. Experimental design Dose–response curves of tramadol-mediated antinociception following systemic (3, 10, 50 mg/kg i.p.) and local (50, 100, 250, 400 μg/paw i.pl.) administration were assessed and the respective ED50 ± standard error values were determined (Tallarida, 2000). To assess if the antinociception following intraplantar tramadol was due to local action, formalin was administered in one paw and the tested drug in the contralateral paw. Moreover to evaluate if the local effect of tramadol underlies an opioid mechanism of action, naloxone (100 μg/paw) was simultaneously injected by the intraplantar pathway, and results were compared to those obtained with an effective peripheral dose of morphine (5 μg/paw). Local doses of naloxone and morphine were chosen from pilot experiments or from previous reports (Mixcoatl-Zecuatl et al., 2000). Since no significant differences were seen between the ED50's for the two phases of the test, subsequent experiments were based upon the ED50 data from the second phase of the formalin test. Thus a dose–response curve was constructed from the concurrent delivery of the two routes of administration in a constant dose ratio of ED50's (local pathway ED50 = 134.6 μg/paw + systemic pathway ED50 = 7.2 mg/kg), and subsequent dilutions thereafter (local ED50/2 + systemic ED50/2, local ED50/4 + systemic ED50/4 and local ED50/8 + systemic ED50/8), as shown in Table 1. In order
Fig. 1. Time-course of the antinociceptive effect of (A) local (peripheral) administration of tramadol 3 (○), 10 (∇) and 50 (▾) mg/kg i.p or saline (●); (B) systemic administration of tramadol 50 (○), 100 (∇), 250 (▾) and 400 (◼) μg/ paw i.pl. or saline (●); and (C) local–systemic combination of tramadol 17.7 (○), 35.4 (∇), 70.8 (▾) and 141.8 (◼) “dose units” (see text for details) or saline (●). Data represent the mean ± S.E.M. of 6 rats.
for nociceptive behavior immediately after formalin injection. Nociceptive behavior was quantified as the number of flinches of the injected paw 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
Fig. 2. Evidence of the significant antinociceptive effect of ipsilateral (IL) but not contralateral (CL) locally administered tramadol (400 μg/paw). This effect is not reversed by naloxone (100 μg/paw) in contrast to an effective dose of morphine (100 μg/paw) in the second phase of the formalin test. Results are presented as the mean of 6 rats (± S.E.M.). ⁎ Denotes differences from the formalin untreated group (p b 0.05).
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(Tallarida, 2000). Briefly, the ED50 values of each route were plotted on the x- and y-axes, respectively. The theoretically additive dose combination was calculated. From the variance of the total dose, individual variances for each route in the combination were obtained. To further describe the magnitude of the interaction, the interaction index (γ) (Tallarida, 2002) was calculated as: ((ED50 dose in combination for route 1))/(ED50 value for route 1 given alone)) + ((ED50 dose in combination for route 2)/(ED50 value for route 2 given alone)). A value of 1 indicates an additive interaction whereas a value b 1 indicates a synergistic interaction and a value N1 implies an antagonistic interaction. Finally to evaluate the opioid component underlying “selfsynergism”, local (100 μg/paw i.pl.) and systemic (1 mg/kg i.p.) naloxone was administered simultaneously with the ED50 of the route combination. Data analysis and statistics Results are presented as the mean ± S.E.M. for 6–8 animals per group. Statistical significance between the theoretical additive point and the experimentally-derived value was evaluated by using Student's t-test. p b 0.05 was considered to indicate a significant synergistic interaction between the two routes of administration (Tallarida, 2000, 2001). Data from the experiments testing the role of the opioid pathway in the mechanism of self-synergism were analyzed using the one way ANOVA followed by the Tukey test. Results Local and systemic antinociceptive effect of tramadol
Fig. 3. Comparative dose–effect curves for the intraperitoneal (A) and intraplantar (B) administration of (±)-tramadol evaluated in the first (open circles) and the second phase (filled circles) of the formalin test. Data are shown as the mean (n = 6) ± S.E.M.
to correctly handle the data from such combinations, a dummy variable of dosing was introduced, namely “dose units” since the constituent dose units, μg/paw (local) and mg/kg (systemic) cannot be mixed, so that “dose units” corresponds to the arithmetical sum of an intraplantar (μg/paw) plus an intraperitoneal (mg/kg) dose combination of tramadol. In order to evaluate the nature of any site–site antinociceptive interaction between the two routes of administration, isobolograms were constructed using doses producing 50% maximum possible effect (ED50) obtained when tramadol was administered for each individual route or combined as previously described
Subcutaneous injection of formalin into the hind paw produced a biphasic pattern of flinching behavior. The first phase started immediately after administration of formalin and then diminished gradually in approximately 10 min. The second phase started at 15 min and lasted until 1 h. Fig. 1(A–C) shows the time-courses of flinching behavior in control and treated rats with increasing doses of tramadol given by local, systemic and both routes. Local ipsilateral, but not contralateral administration of single or dualsite tramadol produced a significant (p b 0.05) reduction in the flinching behavior otherwise observed after formalin injection,
Table 2 Effect of tramadol administered by local (intraplantar) and systemic (intraperitoneal) routes alone and in combination in both phases of the formalin test
Local (μg/paw) Systemic (mg/kg) Theoretical combination (“dose units”) Experimental combination (“dose units”) Interaction index
First phase ED50
Second phase ED50
171.0 ± 44.7 10.2 ± 2.1 422.2 ± 50.5 211.1 ± 13.6⁎ 0.50 ± 0.06
134.6 ± 25.1 7.1 ± 0.5 138.5 ± 9.2 45.9 ± 3.9⁎ 0.33 ± 0.03
ED50: effective dose resulting in a 50% reduction in the nociceptive response. Data are the mean ± S.E.M. ⁎Significantly different from the theoretical combination data (p b 0.05), by the Student's t-test.
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Tramadol was able to produce a dose-dependent suppression of the flinching response in both phases of the formalin test not only after the intraperitoneal but also following the intraplantar dosing, although the antinociception was superior following the systemic administration (Fig. 3A–B). In addition, no differences in the analgesic potency (ED50) of tramadol between the first and second phases of the test were noticed following administration by either route (Table 2). Therefore, the experimental design of the isobolographic analysis was based on the ED50's for the second phase. In order to detect spontaneous central side effects, an assessment of behavioral neurotoxicity was performed as described by Yamamoto and Yaksh (1992). No abnormality of placing, stepping, or righting reflexes was exhibited in the rats before or after treatments in either group: control or treated by any of the studied routes of administration (data not shown). Isobolographic analysis of the dual-site synergism in the antinociceptive effect of tramadol
Fig. 4. Isobolograms of the ED50 values for tramadol administered concurrently by the systemic (intraperitoneal) and local (intraplantar) pathways in the first (A) and the second phase of the formalin test. Horizontal and vertical bars indicate S.E.M. The oblique line between the x- and y-axis is the theoretical additive line. The point in the middle of this line, indicated by “T”, is the theoretical doseadditive point calculated from the separate ED50 values. The point indicated by “E” is the experimentally derived ED50 value observed with the combination. In all cases the experimental point is situated below the additive line, indicating a significant synergism (p b 0.05).
suggesting that the response was actually elicited by local mechanisms (Fig. 2). Moreover this effect was not reversed by naloxone (100 μg/paw) in contrast to an effective dose of morphine (100 μg/paw) in the second phase of the formalin test.
Tramadol was also able to produce a dose-dependent suppression of the flinching response in both phases of the formalin test following the systemic–local route combination at a fixed ratio (Fig. 3C). The isobolographic analysis of the dual-site interaction in the antinociception produced by tramadol given by the two different routes is shown on Fig. 4. In both phases of the formalin test the experimental point lies far below the additive line indicating a significant synergism (p b 0.05). In addition, the experimental ED50 values for the routes in combination were significantly lower than the theoretical ED50 by factors of 2 and 3 in phase 1 and 2 of the test, respectively (Table 2). Moreover, the selfsynergistic interaction between the two routes of administration was confirmed from the estimation of the interaction index, for which the values of 0.5 and 0.3 in phases 1 and 2 of the formalin test were far less than 1 (Table 2).
Fig. 5. The dual-site synergized tramadol antinociception is reversed by systemic (1 mg/kg i.p.; NLXS) or combined local + systemic naloxone (100 μg/paw i.pl. + 1 mg/kg i.p.), but not by local naloxone (100 μg/paw i.pl.; NLXS) in the second phase of the formalin test (B). Neither local (100 μg/paw i.pl.) nor systemic (1 mg/kg i.p.) naloxone by itself modified the natural nociceptive response to the intraplantar injection of formalin (open bars). Results are presented as the mean of 6 rats (±S.E.M.). ⁎p b 0.05 vs formalin untreated group (saline); ⁎⁎p b 0.05 vs the TMDL + TMDS (dual-site tramadol combination) group.
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Role of the opioid pathway in the dual-site synergism of tramadol antinociception In order to evaluate whether peripheral and/or central opioid mechanisms were involved in the dual-site synergism, local (100 μg/paw i.pl.) and systemic (1 mg/kg i.p.) naloxone was administered alone or jointly with the experimental ED50 of the combination (211 and 46 “dose units” for phases 1 and 2 of the formalin test, respectively). Neither local nor systemic naloxone by itself modified the formalin-evoked nociceptive response (Fig. 5A). Local naloxone (100 μg/paw i.pl.) was unable to reverse the increased antinociceptive effect of the combination in the experimental model. In contrast systemic naloxone (1 mg/ kg i.p.) alone or combined with local naloxone partially blocked the increased antinociception of tramadol given by two routes (Fig. 5B) in the second phase of the formalin test. Discussion Management of acute and chronic pain is important, not only for patient well-being but also to prevent long-term complications and morbidity (APSQCC, 1995). Although the analgesic efficacy of tramadol has been shown, side effects, particularly opioidrelated, remain a concern (Silvasti et al., 1999). In order to overcome this, new therapeutic strategies, such as balanced analgesia, have been developed (Kehlet et al., 1999). In the present study we found that the adaptation of the balanced analgesia concept to the combination of multiple routes of administration of a single agent, namely local and systemic tramadol, promises to maintain a good antinociceptive effect with reduced central and systemic exposure to the opioid. There are many descriptions of tramadol analgesia after its systemic administration (Lee et al., 1993; Scott and Perry, 2000), but little evidence regarding the peripheral action of tramadol is available. The latter is important because by limiting the exposure of a drug to the periphery, opioid-mediated central side effects can be markedly reduced. In that sense, although previous findings suggested a role for tramadol as a potential local anesthetic agent (Pang et al., 1998; Altunkaya et al., 2003), to our knowledge this is the first report showing that tramadol is able to produce local antinociception in an experimental model of acute and tonic pain. In this study we found that intraplantar tramadol was able to dose-dependently diminish the nociceptive behavior of flinching in both phases of the formalin test in rats by an unclear mechanism of action. One possible explanation is that tramadol by itself interacts with opioid receptors in the primary afferent. However, several lines of evidence support alternative mechanisms. Indeed, previous findings have shown that other opioid agents produce analgesic effects due to an increased density of opioid receptors on peripheral sensory nerves during painful inflammatory processes (Junien and Wettstein, 1992; Watterson et al., 2004). However, tramadol binding affinity for opioid receptors appears to be too low to account for the antinociceptive effects through this system (Lee et al., 1993). Moreover, it is known that the (+)enantiomer of the active metabolite of tramadol is about 200-fold more potent in activating the μ-opioid receptor than the parent
drug, but no tramadol-metabolizing enzymes are present in the inflamed tissue (Gillen et al., 2000). In addition, local naloxone was unable to block the increased antinociceptive response produced by local or the local–systemic tramadol combination in contrast to that observed with an effective dose of local morphine. Therefore, a mechanism other than opioid peripheral pathway activation must be responsible for the local antinociceptive action of tramadol. In that regard, it was postulated that tramadol has a local anesthetic effect similar to that of lidocaine following intradermal injection (Pang et al., 1998). On the other hand, tramadol appears to have anti-inflammatory properties independent of the arachidonic acid pathway (Bianchi et al., 1999). Once confirmed the noticeable local antinociceptive effect of tramadol, the hypothesis that the concurrent administration of the drug by local and systemic pathways produces an increased antinociceptive response was tested. The experimental design was based upon isobolographic analysis using a fixed ratio (19:1 local–systemic dosing) as this methodology is well-recognized as the standard to evaluate drug combinations (Tallarida, 2000). Our results demonstrated a marked self-synergism in the antinociceptive effect between the two routes of administration, i.e. the observed ED50 of the combination was 2 and 3 times (phase 1 and 2 of the formalin test, respectively), less than the theoretical ED50. These observations imply that systemic exposure to tramadol may be reduced up to 3 times, if combined with local administration (e.g. topical formulation or infiltration in wounds), and still retain its antinociceptive efficacy. We used the formalin test to evaluate the antinociception of the tramadol site–site interaction. The observed biphasic pattern reflects different pathological processes, different sensitivities to centrally and peripherally acting analgesic agents, and perhaps different pain qualities (Abbott et al., 1995). Notwithstanding, we found that the potency (ED50) of tramadol administered by the local or systemic routes was not significantly different between the first and second phases of the formalin test. Therefore, the experimental design of the isobolographic analysis was based on the ED50 for the second phase (Yoon and Yaksh, 1999). However, the evaluation of the opioid contribution to the synergism showed that the mixed local–systemic administration of naloxone was able to completely block the increased analgesic effect in the first phase (data not shown). In contrast, only a partial blocking of the synergism was seen in the second phase of the formalin test. The site–site synergism is a phenomenon that has been demonstrated previously. Indeed, Raffa et al. (2000) demonstrated the dual-site analgesic synergism of acetaminophen following the spinal and supraspinal administration of the drug. The authors proposed that the systemic administration of the drug will lead to the spinal release of an opiod-like compound and would explain the self-synergism of acetaminophen in mice. That is, the finding of synergism may not only identify a potential therapeutic use of a drug combination, but also may illuminate the mechanism of action of a drug mixture and even of a single agent (Tallarida, 2001). In this study, although the actual mechanism underlying the synergism remains unclear, the opioid blockade of the antinociceptive response with systemic, but not local naloxone suggests
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that the opioid pathway is not the only mechanism of the potentiation. Furthermore, it is well-known that tramadol-induced analgesia is not accounted for by a single mechanism of action. In fact, following the systemic administration of tramadol, the parent drug and an active metabolite interact with each other by opioid and monoaminergic pathways to reduce the nociceptive input to the spinal cord (Raffa et al., 1993; Valle et al., 2000; Garrido et al., 2003). Thus, in addition to the previously mentioned local anesthetic and anti-inflammatory properties of local tramadol, other mechanisms may be participating. For instance, it has been recently shown that local antidepressants with monoaminergic actions are able to produce analgesia in experimental models of tonic and neuropathic pain (Sawynok et al., 1999; Esser and Sawynok, 1999). Experiments testing the ability of tramadol to inhibit the reuptake of norepinephrin and serotonin at the periphery (currently underway) might help to elucidate the observed self-synergism. In conclusion, this study illustrated for the first time the local peripheral antinociceptive effect of tramadol in a model of acute pain. Our study is the first to demonstrate self-synergism in antinociception by tramadol when administered by two different routes. The observed site–site interaction provides insights for alternatives in the management of pain. Notwithstanding, in order to clarify the mechanism of tramadol local action, as well as that of synergistic combination, further studies should be conducted. Acknowledgements This study was partially supported by the following grants CONACYT-38940-M, PROMEP-103.5/02/2351 and C0-FAI04-3.4. A.J. Pozos-Guillen and P. Aguirre-Bañuelos are CONACYT fellows. We would like to thank Dr. Paul Kretchmer ([email protected]) at San Francisco Edit for his assistance in editing this manuscript. References Abbott, F.V., Franklin, K.B., Westbrook, R.F., 1995. The formalin test: scoring properties of the first and second phases of the pain response in rats. Pain 60, 91–102. Altunkaya, H., Ozer, Y., Kargi, E., Babuccu, O., 2003. Comparison of local anaesthetic effects of tramadol with prilocaine for minor surgical procedures. British Journal of Anaesthesia 90, 320–322. American Pain Society Quality of Care Committee Quality, 1995. Improvement guidelines for the treatment of acute pain and cancer pain. JAMA 274, 1874–1880. Bamigbade, T.A., Davidson, C., Langford, R.M., Stamford, J.A., 1997. Actions of tramadol, its enantiomers and principal metabolite, O-desmethyltramadol, on serotonin (5-HT) efflux and uptake in the rat dorsal raphe nucleus. British Journal of Anaesthesia 79, 352–356. Bianchi, M., Rossoni, G., Sacerdote, P., Panerai, A.E., 1999. Effects of tramadol on experimental inflammation. Fundamental & Clinical Pharmacology 13, 220–225. Chen, Y., Chan, S.Y., Ho, P.C., 2002. Isobolographic analysis of the analgesic interactions between ketamine and tramadol. Journal of Pharmacy and Pharmacology 54, 623–631. Esser, M.J., Sawynok, J., 1999. Acute amitriptyline in a rat model of neuropathic pain: differential symptom and route effects. Pain 80, 643–653. Garrido, M.J., Sayar, O., Segura, C., Rapado, J., Dios-Vieitez, M.C., Renedo, M.J., Troconiz, I.F., 2003. Pharmacokinetic/pharmacodynamic modeling of the antinociceptive effects of (+)-tramadol in the rat: role of cytochrome P450 2D
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