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Carbachol interactions with nonsteroidal anti-inflammatory drugs
H.F. Miranda, F. Sierralta, and G. Pinardi
Abstract: The inhibition of cyclooxygenase enzymes by nonsteroidal anti-inflammatory drugs (NSAIDs) does not com-pletely explain the antinociceptive efficacy of these agents. It is known that cholinergic agonists are antinociceptive,and this study evaluates the interactions between carbachol and some NSAIDs. Antinociceptive activity was evaluatedin mice by the acetic acid writhing test. Dose–response curves were constructed for NSAIDs and carbachol, adminis-tered either intraperitoneally (i.p.) or intrathecally (i.t.). The interactions of carbachol with NSAIDs were evaluated byisobolographic analysis after the simultaneous administration of fixed proportions of carbachol with each NSAID. Allof the drugs were more potent after spinal than after systemic administration. The combinations of NSAIDs andcarbachol administered i.p. were supra-additive; however, the i.t. combinations were only additive. Isobolographic anal-ysis of the coadministration of NSAIDs and carbachol and the fact that atropine antagonized the synergistic effect sug-gest that carbachol may strongly modulate the antinociceptive activity of NSAIDs; thus, central cholinergic modulationwould be an additional mechanism for the antinociceptive action of NSAIDs, unrelated to prostaglandin biosynthesisinhibition.
Key words: antinociception, nonsteroidal anti-inflammatory drugs, cholinergic, carbachol, writhing test.
Résumé : L’inhibition des cyclooxygénases par les anti-inflammatoires non stéroïdiens (AINS) n’explique pastotalement l’efficacité antinociceptive de ces agents. On sait que les agonistes cholinergiques sont antinociceptifs et laprésente étude évalue les interactions entre le carbachol et certains AINS. L’activité antinociceptive a été évaluée chezdes souris en utilisant le test de contorsions à l’acide acétique. Des courbes dose-réponse ont été établies pour lesAINS et le carbachol, administrés par voie intrapéritonéale (i.p.) ou intrathécale (i.t.). Les interactions carbachol–AINSont été évaluées par l’analyse des isoboles après l’administration simultanée de concentrations fixes de carbachol avecchacun des AINS. Tous les médicaments ont été plus puissants après l’administration spinale qu’après l’administrationsystémique. Les combinaisons AINS–carbachol administrées par voie i.p. ont été supra-additives; toutefois, lescombinaison i.t. ont été uniquement additives. L’analyse des isoboles de la co-administration AINS-carbachol etl’antagonisme de l’effet synergique par l’atropine donnent à penser que le carbachol pourrait moduler fortementl’activité antinociceptive des AINS; ainsi, la modulation cholinergique centrale serait un mécanisme additionnel del’action antinociceptive des AINS, sans rapport avec l’inhibition de la biosynthèse des prostaglandines.
Mots clés : antinociception, anti-inflammatoires non stéroïdiens, cholinergique, carbachol, test de contorsions.
[Traduit par la Rédaction] Miranda et al. 1179
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) produceantinociception by inhibiting cyclooxygenase-1 (COX-1) andcyclooxygenase-2 (COX-2) enzymes (Cyrer and Feldman1998; Smith et al. 2000; Paik et al. 2000). Despite severalstudies that emphasize the COX-mediated antinociceptiveactivity of NSAIDs, the selective inhibition of COX does notexplain all of the antinociceptive efficacy of these agents invarious models of acute pain (Miranda et al. 1993, 2001,2002; Pinardi et al. 2001). Several studies have demonstratedthat cholinergic agonists possess antinociceptive properties
(Eisenach 1999). Thus, antinociceptive effects have been re-ported for carbachol administered either intrathecally(Svensson et al. 1991; Eisenach and Gebhart 1995; Abramand O’Connor 1995; Naguib and Yaksh 1997) ormicroinjected into the periaqueductal gray matter of the rat(Guimaraes and Prado 1994; Guimaraes et al. 2000). Fur-thermore, it has been suggested that acetylcholine is an en-dogenous antinociceptive compound that may act throughmonoaminergic pathways (Gillberg et al. 1989; Iwamoto andMarion 1993; Guimaraes and Prado 1994). On the otherhand, cholinergic agonists exert a positive modulatory actionon opioid and clonidine antinociception (Eisenach andGebhart 1995; Zu et al. 1996; Hood et al. 1997).
Although previous studies have demonstrated thatNSAIDs and cholinergic agents can induce antinociception,the characteristics of the possible interactions between thesedrugs have not been evaluated. The demonstration of syn-ergy or supra-additive interactions between analgesic agentsmay have clinical relevance, because it could provide a ratio-nale for the reduction of analgesic doses in chronic pain
Can. J. Physiol. Pharmacol. 80: 1173–1179 (2002) DOI: 10.1139/Y02-145 © 2002 NRC Canada
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Received 3 September 2002. Published on the NRC ResearchPress Web site at http://cjpp.nrc.ca on 18 December 2002.
H.F. Miranda,1 F. Sierralta, and G. Pinardi. PharmacologyProgram, ICBM, Faculty of Medicine, University of Chile,Independencia 1027, Santiago 7, Chile.
1Corresponding author (e-mail: [email protected]).
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states and, consequently, of associated side effects. The pur-pose of the current study was to examine the interactions be-tween the cholinergic agonist carbachol and several of themost frequently used NSAIDs, using isobolographic analysisand a visceral algesiometric test.
Materials and methods
CF-1 mice weighing 28 ± 2 g were used throughout theexperimental work. The animals were mantained in plasticcages (8 animals/cage) on a 12-h light cycle, at an ambienttemperature of 22 ± 1°C, and were fed and watered ad libi-tum. Mice were bred in our facilities and were acclimatizedto the laboratory environment for at least 2 h before beingused. The animals were cared for in accordance with theguidelines of the Canadian Council on Animal Care and theexperimental protocol was approved by the Committee ofAnimal Use and Care of the Faculty of Medicine, Universityof Chile. In particular, the duration of the experiments wasas short as possible, the number of animals involved waskept to a minimum, and the animals were killed immediatelyafter the recording period by cervical dislocation. Each ani-mal was used only once and received only one dose of thedrugs tested. All drugs were freshly prepared by dissolvingthem in normal saline or in a slightly hyperbaric solution ofglucose (6%). All observations during the assay were per-formed by the authors in a randomized and blinded manner.Evaluation of antinociceptive activity was accomplished aspreviously reported (Miranda et al. 1993). Briefly, intra-peritoneal (i.p.) administration was done by injecting the to-tal dose in a constant volume of 10 mL/kg, 30 min beforethe algesiometric test. Intrathecal (i.t.) administration wasdone with the Hylden and Wilcox (1980) technique, and thetotal dose was injected 15 min before the algesiometric testin a constant volume of 5 �L per 30 g mouse body weight,dissolved in a slightly hypertonic solution of glucose (6%) tolimit diffusion. The procedure was performed rapidly with ahigh degree of accuracy and reproducibility. The times ofdrug administration before the algesiometric test (30 min fori.p. and 15 min for i.t.) were found in previous experimentsto be near the time of onset of the maximum analgesic ef-fect. Control animals (saline or 6% glucose) were inter-spersed concurrently with the drug-treated animals, whichprevented all the controls being run on a single group ofmice at one time during the course of the investigation.
Mice were injected i.p. with 10 mL/kg of 0.6% aceticacid, and the number of writhes was counted during a single5-min period starting 5 min after the administration of theacetic acid solution. A writhe was defined as a contractionof the abdominal muscles accompanied by elongation of thebody and extension of the hindlimbs. Dose–response curves,determined near the time of peak effect, were constructed toassess the antinociceptive actions of the different NSAIDsand carbachol administered either i.p. or i.t.. Eight animalswere used at each of at least four doses to determine a dose–response curve. The dose that produced 50% of antinoci-ception (ED50, 50% reduction of control writhes) was calcu-lated using standard linear-regression analysis of the dose–response curve (Tallarida and Murray 1986). Antinociceptiveactivity was expressed as percent inhibition of the usual
number of writhes observed in i.p. saline (19.8 ± 0.30, n = 58)or i.t. glucose (20.1 ± 0.43, n = 55) control animals.
The interaction of the cholinergic agonist carbachol withthe antinociceptive effects of NSAIDs was evaluated by thesimultaneous administration of fixed proportions of carbacholwith each NSAID and by performing an isobolographicanalysis for the different combinations as described byTallarida et al. (1989). For each drug mixture, the ED50 andits associated 95% confidence interval were determined bylinear-regression analysis of the log dose–response curve(eight animals at each of at least four doses) and comparedby a t test to a theoretical additive ED50 (ED50add) obtainedfrom the following calculation: ED50add = ED50NSAID/(P1 + R ×P2), where ED50NSAID is the ED50 determined for theNSAID, R is the potency ratio of the NSAID alone tocarbachol alone, P1 is the proportion of NSAID, and P2 isthe proportion of carbachol in the total mixture. In the pres-ent study, fixed-ratio proportions were selected by first com-bining the ED50 of each compound and then constructing adose–response curve in which ED50 fractions (1:2, 1:4, 1:8,and 1:16) of carbachol–NSAID combinations were adminis-tered. In the equation above, ED50add is the total dose, andthe variance of ED50add was calculated from the fraction ofthe ED50 values (i.e., 0.5) in the combination as follows:variance of ED50add = (0.5)2 × variance of ED50NSAID +(0.5)2 × variance of ED50carbachol (Pinardi et al. 2001). Fromthese variances, confidence limits were calculated and re-solved according to the ratios of the individual drugs in thecombination. Supra-additivity or synergistic effect is definedas the effect of a drug combination that is higher and statisti-cally different (i.e., ED50 significantly lower) than the theo-retical calculated equivalent effect of a drug combinationwith the same proportions. When the drug combination givesan experimental ED50 not statistically different from the the-oretically calculated ED50, the combination has an additiveeffect. Additivity means that each constituent contributes itsown potency, and the less potent drug is acting as though itis merely a diluted form of the other (Tallarida 2001). Atro-pine (1 mg/kg, i.p.) was used as an antimuscarinic agent toevaluate the contribution of central cholinergic system mod-ulation on the effect of the interaction of NSAIDs andcarbachol.
Diclofenac, ketoprofen, piroxicam, and meloxicam wereprovided by local laboratories. Carbachol (carbamylcholinechloride) and atropine sulphate were purchased from SigmaChemical Co. (St. Louis, Mo.).
Results are presented as ED50 values with 95% confidencelimits or as means ± SE, according to Tallarida and Murray
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ED50 (mg/kg)
Drug i.p. i.t.
Meloxicam 6.50 (4.95–8.47) 0.21 (0.18–0.25)Diclofenac 7.20 (3.95–13.3) 0.43 (0.41–0.45)Piroxicam 8.50 (6.50–11.2) 0.50 (0.43–0.60)Ketoprofen 30.30 (24.5–37.6) 0.82 (0.73–0.92)Carbachol 0.013 (0.01–0.016) 0.000075 (0.00005–0.00012)
Table 1. ED50 values (with 95% confidence limits) for theantinociceptive effects of nonsteroidal anti-inflammatory drugsand carbachol administered i.p. or i.t.
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(1986). Student’s t test for independent means was used toassess statistical significance (P < 0.05).
Results
Antinociceptive effectsAnimals tested with the different doses of NSAIDs or
carbachol did not exhibit significant visual behavioral ormotor dysfunction. The i.p. or i.t. administration ofcarbachol and the different NSAIDs produced dose-dependent antinociceptive effects in the algesiometric assayof acetic-acid-induced writhes with different relative poten-
cies. The dose–response curves obtained were characterizedby equal efficacy, and the corresponding ED50 values withtheir 95% confidence limits are shown in Table 1. The rankof order of relative potency of NSAIDs after i.p. or i.t. ad-ministration, defined by the ratio of ED50 values in milli-grams per kilogram, was found to be as follows: meloxicam >diclofenac > piroxicam > ketoprofen.
Interactions of NSAIDs and carbacholThe antinociceptive activity induced after the i.p or i.t.
coadministration of fixed ratios of ED50 fractions of NSAIDsand carbachol was examined by the analysis of the
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ED50 (mg/kg)
Drug combinations Theoretical additive, i.p. Experimental, i.p. Theoretical additive, i.t. Experimental, i.t.
Meloxicam and carbachol 3.25 (2.59–2.93) 2.42 (2.18–4.86)* 0.14 (0.11–0.17) 0.18 (0.15–0.20)Diclofenac and carbachol 3.60 (1.84–7.06) 1.64 (1.40–1.92)* 0.22 (0.16–0.27) 0.24 (0.21–0.28)Piroxicam and carbachol 4.37 (2.96–6.46) 1.96 (1.81–2.13)* 0.28 (0.21–0.37) 0.24 (0.21–0.26)Ketoprofen and carbachol 15.15 (10.46–21.94) 8.81 (7.68–10.11)* 0.47 (0.37–0.59) 0.50 (0.42–0.60)
Note: The ratio between the nonsteroidal anti-inflammatory drugs and carbachol in the combinations corresponds to the ratio of the respective ED50s.(See Materials and methods). *, P < 0.05 between theoretical and experimental values.
Table 2. Theoretical additive and experimental antinociceptive ED50 values (with 95% confidence limits) for combinations ofnonsteroidal anti-inflammatory drugs and carbachol administered i.p. and i.t.
Fig. 1. Isobolograms of the simultaneous administration of meloxicam and carbachol administered i.p. (A) and i.t. (C), and ofpiroxicam and carbachol administered i.p. (B) and i.t. (D). Ordinates of i.p. and i.t. administrations are on different scales. �, Theoret-ical additive ED50 of the combination with 95% confidence limits; �, experimental ED50 with 95% confidence limits.
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corresponding isobolograms. The ED50 values for the combi-nations and their 95% confidence intervals are shown inTable 2. All the combinations of NSAIDs and carbachol ad-ministered i.p. provided synergistic or supra-additive effects(Figs. 1A, 1B, 2A, and 2B), since the experimental point ob-tained differed significantly from the theoretically calculatedadditive point (Table 2). All of the combinations of NSAIDsand carbachol administered i.t. were only additive (Figs. 1C,1D, 2C, and 2D).
Effects of atropine on the NSAID–carbacholinteractions
The pretreatment of mice with 1 mg atropine/kg (i.p.) didnot affect the antinociceptive effect of NSAIDs, but it com-pletely antagonized the synergistic or supra-additive effectsof all combinations of NSAIDs and carbachol administeredi.p. (Fig. 3). Pretreatment with atropine did not induce visi-ble behaviour or motor dysfunction.
Discussion
The present work found that i.p. or i.t. administration ofthe different NSAIDs and carbachol inhibit nociception inthe acetic-acid writhing test in mice. This algesiometric test,in which these compounds show a potent antinociceptive ac-
tivity (Miranda et al. 2001, 2002; Pinardi et al. 2001), maybe considered as a model of clinically relevant pain in hu-mans (Reichter et al. 2001). The antinociceptive activity wasdose-dependent and the agents were more potent after spinalthan systemic administration. Since all drugs administeredi.t. displayed a rapid onset of action, the difference in rela-tive potencies does not seem to be due to pharmacokineticfactors, such as the effect of the blood barrier, protein bind-ing (since, in the cerebrospinal fluid, the levels of proteinare low), or rapid metabolism, which are probably more sig-nificant after i.p. administration. In addition, the presentstudy seems to demonstrate that after i.t. administration,which is characterized by a powerful effect upon spinal no-ciceptive processing (Malmberg and Yaksh 1992), NSAIDsand carbachol do not reach significant systemic concentra-tions, since i.t. coadministration of NSAIDs and carbacholdisplayed a significantly different interaction than i.p.coadministration of the same drugs. Moreover, if the spinaleffects of coadministered drugs were mediated by anonspinal redistribution, it would be anticipated that no dif-ference in the type of interaction would be found.
Cholinergic central antinociception results from the acti-vation of nicotinic or muscarinic mechanisms and dependson which site in the central nervous system is activated by acholinergic agonist such as carbachol, which has nicotinic
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Fig. 2. Isobolograms of the simultaneous administration of diclofenac and carbachol administered i.p. (A) and i.t. (C) and ketoprofenand carbachol administered i.p. (B) and i.t. (D). Ordinates of i.p. and i.t. administrations are on different scales. �, Theoretical addi-tive ED50 of the combination with 95% confidence limits; �, experimental ED50 with 95% confidence limits.
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and muscarinic agonist activities (Green and Kitchen 1986;Iwamoto and Marion 1993). At the central nervous systemlevel, cholinergic antinociception seems to be mediated byactivation of M1 postsynaptic muscarinic receptors, even ifM2 muscarinic autoreceptors can also regulate pain by mod-ulating acetylcholine release via a negative-feedback mecha-nism (Ghelardini et al. 1990; Bartolini et al. 1992). Inaddition, the central antinociception induced by cholinergicagonists seems to be mediated in part by the subsequent ac-tivation of spinally projecting noradrenergic neurons, lo-cated in the pontine A7 catecholamine cell group, thatinhibit spinal nociceptive transmission (Nuseir et al. 1990).
Spinal mechanisms of cholinergic antinociception arecomplex, but to explain the interactions with otherneurotransmitters, the existence of muscarinic interneuronsin the pain transmission pathway and (or) of muscarinic de-sensitizing effects on peripheral nociceptors have been pos-tulated (Hartvig et al. 1989; Bernardini et al. 2001). Theactivaction of muscarinic sites in the spinal cord may resulteither in inhibition of the activity of nociceptive dorsal-hornneurons or in reduced transmitter release from small-diameter nociceptive primary afferents in the dorsal horn, orboth (Gillberg et al. 1989). In addition, the existence ofmuscarinic receptor subtypes (ACh-M1, ACh-M2, and ACh-M3) in the dorsal horn of the spinal cord has been demon-
strated by autoradiographic, binding, and pharmacologicalstudies (Gillberg and Askmark 1991; Iwamoto and Marion1993; Naguib and Yaksh 1997). It has been suggested thatantinociceptive responses produced by intrathecally adminis-tered cholinergic agonists involve the stimulation ofmuscarinic M1 and (or) M2 cholinergic receptors (Eisenach1999) and may also involve subsequent activation of �2-adrenergic and 5-HT1C, 5-HT2, and 5-HT3 serotonergic re-ceptor systems at the level of the spinal cord (Gillberg et al.1989). Furthermore, other mechanisms, such as hyper-polarization of neurons, reduction in the release ofpronociceptive neurotransmitters, and activation of the nitricoxide – cGMP pathway have been suggested as mediators ofcholinergic antinociception through elevation of endogenousacetylcholine (Guimaraes and Prado 1999).
The synergism obtained after the systemic administrationof the cholinergic agonist carbachol with all the NSAIDstested supports the relevance of supraspinal antinociceptionmodulation produced by the activation of muscarinic or nic-otinic receptors, with activation of descending inhibitorynoradrenergic and serotonergic pathways. This activationpresumably occurs by modulating cholinergic interneuronsin relay stations through which descending pathways fromthe periaqueductal gray project to the spinal cord (Gillberget al. 1989; Iwamoto and Marion 1993; Guimaraes and
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Fig. 3. Effects of atropine (1 mg/kg, i.p.) on the number of writhes induced in 5 min by the administration of the NSAID alone andby the simultaneous administration of ED50 combinations. Meloxicam (MELO) and carbachol (CCh) (A); diclofenac (DIC) and CCh(B); piroxicam (PIRO) and CCh (C); ketoprofen (KETO) and CCh (D). Each bar represents the mean ± SE of at least 8 animals. *,P < 0.05 with respect to NSAID and carbachol.
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Prado 1994, 1999; Guimaraes et al. 2000). This is supportedby the antagonistic effect of atropine, a paradigmatic anti-muscarinic agent that crosses the blood–brain barrier, on theantinociceptive effect of the combinations of i.p.-administeredNSAIDs and carbachol. NSAID-induced antinociception wasnot affected by atropine; however, atropine completely an-tagonized the antinociceptive effects of the combinations ofi.p. NSAIDs and carbachol. These observations suggest thatNSAIDs and carbachol induce antinociception by unrelatedmechanisms. Synergism or supra-additivity would be ex-pected if two analgesic drugs act through different mecha-nisms, since the effect on the same mechanism wouldproduce additive interactions (Pinardi et al. 2001).
Synergistic mechanisms between drugs may be influencedby pharmacodynamic interactions (at the receptor or second-messenger levels) and by functional pharmacokinetic inter-actions due to different activity at diverse anatomic sites(Solomon and Gebhart 1994). These considerations mightexplain the differences in relative potency obtained with thecoadministration of carbachol and the different NSAIDs.
The differences obtained between i.p. and i.t. coadmin-istration of carbachol with NSAIDs are in agreement withprevious works, in which synergistic antinociceptive interac-tions were obtained after systemic but not i.t. administrationof analgesic drugs, since systemically administered drugsreach both supraspinal and spinal sites, and i.t. drugs havelimited diffusion and a more local effect (Roerig et al. 1988;Solomon and Gebhart 1994; Miranda et al. 2002). It hasbeen suggested that drug interactions may involve the activa-tion of different pathways of pain inhibition; thus, systemicadministration may stimulate descending pain inhibitorypathways usually mediated by noradrenaline and serotonin,while these inhibitory pathways may not be activated to thesame degree by i.t. administration (Roerig et al. 1988; Suhand Tseng 1988).
The expression of constitutive COX-1 and COX-2 in thecentral nervous system has been reported (Yermakova et al.1999; Niwa et al. 2000). The inhibitory effects of NSAIDson these enzymes could undoubtely contribute to their anti-nociceptive action; however, cholinergic mechanisms relatedto COX inhibition in the central nervous system have notbeen described. Consequently, the synergy reported in thepresent work suggests that cholinergic modulation of theantinociceptive activity of NSAIDs may be an additionalmechanism of action of these drugs, unrelated to prostaglan-din synthesis inhibition, which contributes to their analgesiceffect.
Acknowledgements
This work was supported by Project No. 1990842,Fundación para el Desarrollo de Ciencia y Tecnología(FONDECYT Chile). The expert technical assistance ofJ. López and A. Correa is gratefully acknowledged.
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