Anesth Analg 2001;93:430-435
© 2001 International Anesthesia Research Society
PAIN MEDICINE
An Isobolographic Analysis of the Adrenergic Modulation of Diclofenac Antinociception
Hugo F. Miranda, PhD,
Fernando Sierralta, DDS, and
Gianni Pinardi, MD
Pharmacology Program, ICBM, Faculty of Medicine, University of Chile, Santiago, Chile
Address correspondence and reprint requests to H. F. Miranda, PhD, Pharmacology Program, PO Box 70.000, Santiago 7, Chile. Address e-mail to hmiranda{at}machi.med.uchile.cl
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Abstract
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We evaluated the noradrenergic modulation of the antinociceptive activity of diclofenac in mice using the acetic acid writhing test. Dose-responsecurves were obtained for the antinociceptive effect of diclofenac, phenylephrine, clonidine, desipramine, prazosin, and yohimbine administered both systemically and intrathecally, and ED50s were calculated. Noradrenergic modulation was evaluated by performing an isobolographic analysis of the systemic or intrathecal coadministration of fixed-ratio combinations of diclofenac with each adrenergic drug. The systemic, but not the intrathecal, combinations of diclofenac with phenylephrine or clonidine showed supraadditivity, suggesting that the activation of 
1 and 2 adrenoceptors interfered with the nociceptive transmission at spinal and supraspinal levels. Supraadditive effects were not demonstrated for the intrathecal injection of diclofenac combined with phenylephrine, clonidine and a selective norepinephrine uptake inhibitor (desipramine) or adrenergic antagonists. We conclude that interaction between adrenoceptors and diclofenac can modulate antinociception by activating common or different mechanisms. Diclofenac has an antinociceptive activity that, in addition to cyclooxygenase inhibition, can be modulated by additive and supraadditive interactions with adrenergic drugs.
IMPLICATIONS: Diclofenac analgesia in mice can be modulated by interaction with adrenergic drugs. The systemic but not the intrathecal administration of phenylephrine and clonidine produced supraadditive interactions. For desipramine, prazosin, and yohimbine, supraadditive interactions were not statistically demonstrated. The coadministration of drugs inducing supraadditive effects could be clinically relevant for the treatment of chronic pain because of reduction of doses and side effects.
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Introduction
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Diclofenac (DIC) is a nonsteroidal antiinflammatory drug with potent antiinflammatory and antinociceptive activity. Antinociception by nonsteroidal antiinflammatory drugs has traditionally been attributed to peripheral tissue cyclooxygenase inhibition with inhibition of prostaglandin biosynthesis. However, the antinociceptive effect seems to have a central nervous component observed after visceral noxious stimuli, which probably indirectly involves the opioid system, the N-methyl-D-aspartate receptor, and the nitric oxide generating system, and which is reduced by the descending 5-hydroxytryptamine modulation of nociceptive transmission at the spinal level (1–5). In addition to its ability to block cyclooxygenase, DIC has a direct effect on hyperalgesia that seems to be independent of central or peripheral opioid effects (6). The antinociceptive action of DIC could thus be accounted for by several peripheral and/or central mechanisms besides the inhibition of cyclooxygenase, such as actions on neurotransmitters or neuromodulators involved in the nociceptive system, specifically regarding catecholaminergic and serotonergic functions (7).
Noradrenergic neurons descending through the dorsal lateral funniculus from the brainstem to the dorsal horn significantly contribute to the modulation of pain by controlling impulse transmission (descending inhibitory pathway). -Adrenergic agonists, such as clonidine, possess significant antinociceptive activity by a central action on the brainstem and a spinal action on the substantia gelatinosa of the dorsal horn (8,9). However, the influence of noradrenergic systems on the mechanism of action of DIC has not been extensively studied and the extent of noradrenergic modulation of DIC analgesia is not completely understood. In the present work, the modulation of the antinociceptive effects of DIC by adrenergic mechanisms was studied in mice by using a chemical model of visceral pain to evaluate if the known antinociceptive activity of adrenergic drugs can act synergically with the effect of DIC.
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Materials and Methods
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CF-1 mice weighing 20–25 g obtained from the Instituto de Salud Pública of Chile were used throughout the experimental work. The animals were acclimatized to the laboratory environment for at least 2 h before being used and the ethical standards guidelines were followed as previously described (10). In particular, the duration of the experiments was as short as possible, the number of animals involved was kept to a minimum, and the animals were killed immediately after the recording period by the administration of an anesthetic overdose. Each animal was used only once and received only one dose of the drugs tested. All drugs were freshly prepared by dissolving them in normal saline, and doses were calculated on the basis of the drug salts.
Evaluation of antinociceptive activity was accomplished as previously reported (10). Briefly, intraperitoneal (IP) administration was accomplished by injecting the total dose in a constant volume of 10 mL/kg 30 min before the algesiometric test. For intrathecal (IT) administration, the Hylden and Wilcox (11) technique was used and the doses of the drugs were injected 15 min before the algesiometric test in a constant volume of 5 µL dissolved in a slightly hyperbaric solution of glucose (6%) to limit diffusion. The procedure was performed rapidly with a high degree of accuracy and reproducibility. These times were found in previous experiments to be near the time of onset of maximum analgesic effect. For the algesiometric test, mice were injected IP with 10 mL/kg of 0.6% acetic acid and the number of writhes was counted during a 5-min period starting 5 min after the administration of acetic acid solution. A writhe was defined as a contraction of the abdominal muscles accompanied by an elongation of the body and extension of the hindlimbs. All observations during the assay were performed by the authors in a randomized and blind manner. Control animals (saline) were run interspersed concurrently with the drug-treated animals, which prevented all the controls being run on a single group of mice at one time during the course of the investigation.
Dose-response curves, determined at the time of peak effect, were constructed to assess the antinociceptive action of DIC given by the different routes. Eight animals were used at each of at least four doses to determine a dose-response curve. The dose that produced 50% of antinociception (ED50, 50% reduction of control writhes) was calculated using standard linear regression analysis of the dose-response curve. Antinociceptive activity was expressed as percent inhibition of the usual number of writhes observed in IP saline (24.3 ± 1.0, n = 30) or IT glucose (23.9 ± 0.5, n = 25) control animals. Drugs and control solutions were administered IP (10 mL/kg), or IT (5 µL per mice).
To evaluate the adrenergic modulation of the antinociceptive effect of DIC, phenylephrine (PhE, selective 1-adrenoceptor agonist), prazosin (PRA, selective 1-adrenoceptor antagonist), clonidine (CLON, selective 2-adrenoceptor agonist), yohimbine (YOH, selective 2-adrenoceptor antagonist), and desipramine (DES, selective inhibitor of norepinephrine neuronal uptake) were used. All these drugs exhibited antinociceptive activity when administered IP or IT, and dose-response curves for their effect were constructed as above. The interaction between DIC and the adrenergic drugs was evaluated by simultaneous administration of fixed ratios of DIC with PhE, PRA, CLON, YOH, or DES and performing an isobolographic analysis for the different combinations as described by Tallarida et al. (12). The isobologram was constructed by connecting the ED50 of DIC plotted on the abscissa with the ED50 of the combined drug plotted on the ordinate to obtain the additivity line. For each drug mixture, the ED50 and its associated 95% confidence intervals were determined by linear regression analysis of the dose-response curve (8 animals at each of at least 4 doses) and compared by a t-test to a theoretical additive ED50 obtained from the calculation
equation
where R is the potency ratio of DIC alone to the combined drug alone, P1 is the proportion of DIC, and P2 is the proportion of the combined drug in the total mixture (13). In the present study, fixed-ratio proportions were selected by first combining a dose approximating the ED50 of DIC and each adrenergic compound and then constructing a dose-response curve in which each drug was present in sub-ED50 doses and testing them as described.
The following drugs were used: DIC, a gift from Novartis (Chile); PhE hydrochloride, PRA hydrochloride, CLON hydrochloride, YOH hydrochloride, and DES hydrochloride purchased from RBI (Natick, MA).
Results are presented as mean values ± SEM or as ED50 values and 95% confidence intervals and were examined by analysis of variance followed by Student’s t-test for unpaired data. Parallelism of the lines obtained by linear regression analysis of the dose-response curves was tested according to Tallarida and Murray (14). Significance was accepted at the 0.05 level.
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Results
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The systemic or IT administration of DIC resulted in a dose-dependent antinociceptive activity (Fig. 1). The dose-response curves were parallel, with a relative potency of DIC IT/DIC IP of 18.1 (95% confidence interval, 8.8–34.9). The ED50s for IP administration and for IT injection appear in Table 1.
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Figure 1. Dose-response curves of the antinociceptive effect of diclofenac administered intraperitoneally (IP) and intrathecally (IT) in the acetic acid writhing test of the mouse.
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Table 1. ED50 Values and 95% Confidence Limits for the Antinociception Induced by Different Drugs in the Writhing Test of the Mouse
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The isobolographic analysis of the IP and IT coadministration of DIC with PhE and CLON is shown on Figure 2. Ratios and theoretical and experimental ED50 values for the combinations are given in Table 2. The isobologram of DIC + PhE IP (ratio, 1000:1) showed that the mixture resulted in a supraadditive interaction. The simultaneous injection of DIC + CLON in a fixed proportion of 17400:1 also gave an experimental point lying in the supraadditive region. When the combinations of DIC + PhE (ratio, 4350:1) and DIC + CLON (ratio, 12,000:1) were administered IT, the experimental points denoted additive interactions because supraadditivity was not statistically demonstrated. The isobolograms for the combinations DIC + DES given IP (ratio, 10.5:1) and IT (ratio, 29:1) also resulted in additive interactions (Fig. 3).
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Figure 2. Isobolograms for the intraperitoneal (IP) and intrathecal (IT) co-administration of diclofenac with adrenergic agonists. A, diclofenac + phenylephrine, IP; B, diclofenac + phenylephrine, IT; C, diclofenac + clonidine, IP; D, diclofenac + clonidine, IT. Open circles correspond to the experimental point with 95% confidence limits, and filled circles correspond to the theoretically calculated additive point. The confidence intervals for this point have been omitted for the sake of clarity and appear in Table 2. *P < 0.05 between points (supraadditive interaction).
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Table 2. Theoretical and Experimental ED50 Values with 95% Confidence Limits for Combinations of Diclofenac (DIC) with Adrenergic Drugs
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Figure 3. Isobolograms for the coadministration of diclofenac with desipramine. A, diclofenac + desipramine, IP; B, diclofenac + desipramine, IT. Open circles correspond to the experimental point with 95% confidence limits, and filled circles correspond to the theoretically calculated additive point. The confidence intervals for this point have been omitted for the sake of clarity and appear in Table 2.
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The systemic administration of DIC + PRA (ratio, 5.7:1) and DIC + YOH (ratio, 13:1) was simply additive when analyzed by isobolograms (Fig. 4). The same result was obtained when the mixtures DIC + PRA (ratio, 4.5:1) and DIC + YOH (ratio, 10.5:1) were administered IT. In Table 2, ratios and theoretical and experimental ED50 values for these combinations are listed.
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Figure 4. Isobolograms for the intraperitoneal (IP) and intrathecal (IT) coadministration of diclofenac with adrenergic antagonists. A, diclofenac + prazosin, IP; B, diclofenac + prazosin, IT; C, diclofenac + yohimbine, IP; D, diclofenac + yohimbine, IT. Open circles correspond to the experimental point with 95% confidence limits, and filled circles correspond to the theoretically calculated additive point. The confidence intervals for this point have been omitted for the sake of clarity and appear in Table 2.
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Discussion
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Monoamines play a key role in the modulation of nociception at all levels of the neuraxis, such as in peripheral nociceptive fibers in interaction with sympathetic terminals, in the processing of afferent nociceptive information in the dorsal horn of the spinal cord, in the thalamus integration of nociceptive information, and in higher limbic and cortical integration structures (15). The modulation of the flow of nociceptive information in the spinal cord constitutes a first stage in the processing of pain signals and is accomplished mainly by adrenergic and serotonergic systems originating in the periaqueductal gray, nucleus reticularis paragigantocellularis, and nucleus of the raphe magnum (16).
In the present work, the modulation exerted by adrenergic mechanisms on the antinociceptive effect of DIC was evaluated. The compound administered either systemically or IT produced dose-dependent antinociception in the writhing test, and the dose-response curves were statistically parallel, with DIC being relatively more potent IT than systemically. The selective 1 and 2 adrenergic agonists, PhE and CLON, interacted supraadditively with DIC when given IP, suggesting activation of a central mechanism for DIC antinociception in concordance with the results of Bjorkman et al. (2). The fact that adrenergic agonists interact with DIC by supraadditive mechanisms suggests the activation of different and complementary mechanisms of antinociception because the activation of common mechanism should presumably produce an additive interaction. PhE and CLON are effective antinociceptive drugs in several algesiometric tests and the synergy between adrenoceptor agonists and DIC can be explained by the enhanced antinociception induced in different algesiometric tests by 1 and 2 adrenergic receptors, which is related to the activation of the descending noradrenergic system (16–18). Furthermore, a supraadditive effect was not demonstrated for the IP and IT administration of combinations with DES, a selective noradrenergic neuronal uptake inhibitor that modulates pain sensation by increasing neurotransmitter concentration in the synaptic cleft (19), suggesting modulation of a common mechanism of action. Two different antinociceptive drugs would be expected to be additive if they acted by a common mechanism of action.
The antinociceptive activity of the selective 1-adrenoceptor antagonist PRA has been reported (13,20,21), indicating that 1-adrenoceptors are activated during the nociceptive transmission. However, even if this effect is not completely understood, it seems possible that blocking of the postsynaptic 1-adrenoceptors in the primary afferent neurons might interfere with the nociceptive impulse transmission. Patch-clamp techniques have indicated that 1 receptors in the central nervous system are located postsynaptically and have depolarizing functions, whereas 2 receptors have hyperpolarizing properties and may be located both pre- and postsynaptically (22). It is conceivable that the blockade of 1 receptors might interfere with the depolarization of nociceptive neurons and that hyperpolarization resulting from stimulation of 2 receptors might explain the effect of adrenergic agonists such as CLON.
The administration of IP or IT YOH induced a consistent dose-dependent antinociception in the writhing test. This is in agreement with reports that IT YOH inhibited nociception in the hot plate test and formalin test (21,23) and enhanced tramadol-induced antinociception in the writhing test in mice (24). No synergy between the effect of -adrenergic antagonists and the antinociceptive action of DIC was observed in the present work, the interaction being simply additive in every case, disregarding the route of administration. The mechanisms of the antinociceptive actions of -adrenergic antagonists are complex and not completely clear, and existing knowledge is not sufficient to build a model for the role of these drugs in spinal nociceptive processing. The results of the present work support the conclusion that DIC has a central antinociceptive activity that can be potentiated by noradrenergic receptor systems at supraspinal levels.
The coadministration of drugs inducing analgesic supraadditive effects constitutes a valid approach to the treatment of chronic pain (25), where a reduction of individual doses, and consequently of side effects, could be very important.
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Acknowledgments
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Supported, in part, by FONDECYT Project Number 1990842.
The authors wish to thank J. López and A. Correa for expert technical assistance.
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Accepted for publication April 18, 2001.
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Abstract
Introduction
