Interactions in the antinociceptive effect of tramadol in mice: an isobolographic analysis

European

Journal

of Pain (1998) 2: 343-350

Interactions in the antinociceptive tramadol in mice: an isobolographic

effect of analysis

Gianni Pinardi, Teresa Pelissier and Hugo F. Miranda Department

of Pharmacology,

Faculty

of Medicine,

University

of Chile

Tramadol is a widely-used analgesic for pre- and post-operative pain which has a different pharmacological profile to that of classical opioids, since it does not induce respiratory depression, constipation, sedation, tolerance or dependence. However, tramadol frequently produces nausea and vomiting as side-effects. In the present study, the interactions between tramadol and several adrenergic and serotonergic compounds with antinociceptive activity were studied by isobolographic analysis. Antinociceptive activity was evaluated using the acetic acid writhing test in mice. Dose-response curves for the antinociceptive effect of tramadol, prazosin, clonidine, xylamine, clomipramine and cyproheptadine were obtained, and ED,,s were calculated for isobolographic analysis, which was performed by administration of fixed-ratios of tramadol with each of these drugs, given both systemically and intrathecally. The isobolograms of all combinations tested, either systemically or intrathecally showed superadditivity. The synergies observed with these combinations suggest a complex modulation of the descending noradrenergic and serotonergic systems that exert inhibitory influences on the transmission of nociceptive information, probably in addition to effects on receptors in the primary neurons of the spinal cord. The co-administration of analgesic drugs that produce superadditive effects constitutes a significant new avenue for the treatment of pain, since a similar level of antinociception can be obtained with considerable reductions in the dose of each analgesic

INTRODUCTION The development of drugs for pain treatment is one of the major objectives of pharmacology and medicinal chemistry: different new molecules are synthesizedin order to achieve this aim. Between the new types of molecules, a compound derived from cyclohexanol HCl, named tramadol (TRAM) was developed, and it has been confirmed as an effective and safe analgesic for the management of pain when administered either orally, intramuscularly, intravenously or by patient-controlled analgesia (Budd, 1994; Lehmann, 1994; Sunshine, 1994).TRAM is formulated as a racemic mixture and consistsof 50% (+) and 50% (-) enantiomers. Each enantiomer Paper received 12 April 1998 and accepted in revised form 2 September 1998. G. Pinardi, Department of Correspondence to: Pharmacology, P.O. Box 70 000, Santiago 7, Chile. 1090-3801/98/040343 0 1998 European

+ 08 $12.0010 Federation of Chapters

of the International

possessesdifferent pharmacological properties; (+) tramadol activates opioid receptors and inhibits serotonin uptake, whereas (-) tramadol is a preferential noradrenaline uptake inhibitor; both stereoisomerspotentiate each others antinociceptive effect (Driessen and Reimann, 1992; Raffa et al., 1992). It has been shown that the active metabolite of TRAM, O-desmethyl-tramadol, possessa high affinity for y-opioid receptor subtype and produces a higher analgesic effect than TRAM (Hennies et al., 1988; Friderichs and Becker, 1991;Friderichs et al., 1992; Poulsen et al., 1996). Experimental and clinical trials demonstrated that TRAM displayed a good analgesic efficacy and possesseda potency comparable to codeine (Lehmann, 1994; Sunshine, 1994; Gron et al., 1995). In acute post-operative pain, its efficacy seemsto be similar to that of morphine and, in addition, long-term TRAM treatment in patients Association

for the Study

of Pain

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G.

with nociceptive, neurogenic or sympathogenic pain was as effective as sustained released morphine (Budd, 1994; Vickers and Paravicini, 1995). The antinociceptive activity of TRAM is associated with some properties that configure a pharmacological profile different to that of prototypical or classical opioids. For example, tolerance and dependence were not described after repeated administration of TRAM (Richter et al., 1985; Preston et al., 199 1; Budd, 1994) and TRAM has not been associated with significant opioid side-effects, such as respiratory depression, constipation or sedation (Lehmann, 1994; Vickers et al., 1992). However, TRAM administration produces some inconvenient effects, such as nausea and vomiting, which are typically observed with opioids (Mattia et al., 1993; Gron et al., 1995; Vickers and Paravicini, 1995). Systemicallyand intrathecally-administered drugs may interact to produce different effects, ranging from simple additivity to synergy. For example, synergy between clonidine and opioid receptor subtypes agonists (morphine, fentanyl, meperidine, U-69593, DPDPE) has been described (Ossipov et al., 1990a, b). The present study was designed to investigate, using isobolographic analysis, the interactions between TRAM and several drugs that possess antinociceptive activity and which, by its mechanisms of action may modulate TRAM-induced analgesia. Synergic interactions may have clinical importance, since the total dose of TRAM may be significantly reduced and, consequently, sideeffects should be minimized.

MATERIAL

AND METHODS

CF-1 mice, weighing 2&25 g, 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 (Miranda et al., 1993). 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 European

Journal

of Pain

(1998),

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PINARDI

HAL.

drugs tested. All drugs were freshly prepared by dissolving them in normal saline and doses were calculated on the basis of the drug salts. All observations during the assay were performed by the authors in a randomized and blind manner. Evaluation of antinociceptive activity was accomplished as previously reported (Sierralta et al., 1996). Briefly, intraperitoneal (i.p.) administration was accomplished by injecting the total dose in a constant volume of lOml/kg, 30 min before the algesiometric test. For Intrathecal (i.t.) administration, the Hylden and Wilcox technique (1980) was used and the drugs were injected in a constant volume of 5 ~1. The procedure was performed rapidly with a high degree of accuracy and reproducibility, 15 min before the algesiometric test. 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 i.p. with lOml/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 forelimbs. 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 in order to assess the antinociceptive action of TRAM, prazosin (PRA), clonidine (CLON), xylamine (XYL), clomipramine (CLOMI) and cyproheptadine (CYPRO). Eight animals were used at each of four doses to determine a doseeresponse curve. The dose that produced 50% of antinociception (ED,,: 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 i.p. (27.2 + 1.0, y1=30) or i.t. (26.3) 1.2, n =25) saline control animals. Drugs and saline control solutions were administered i.p. (lOml/kg) or i.t. (5 ~1 per mice), as previously described (Miranda et al., 1993). The interaction of TRAM with the above mentioned drugs was evaluated by simultaneous ad-

ls0130~0GRA~~lc

ANALYSIS

OF TRAMADOL

INTERACTIONS

ministration of fixed ratios of tramadol with: PRA (cz,-adrenoceptor antagonist), CLON (Qadrenoceptor agonist), XYL (selective irreversible inhibitor of norepinephrine neuronal uptake), CLOMI (selective inhibitor of serotonin neuronal uptake) and CYPRO (5-HTz antagonist) and performing an isobolographic analysis for the different combinations as described by Tallarida et al. (1989). The isobologram was constructed by connecting the EDs0 of TRAM plotted on the abscissa with the ED,, of the combined drug plotted on the ordinate to obtain the additivity line. For each drug combination, the EDso and its associated 95% confidence intervals were determined by linear regression analysis of the dose-response curve (eight animals at each of four doses), and compared by a t-test to a theoretical additive EDxO obtained from the calculation: EDso add= EDsoTRAM (Pl + R*P2), where R is the potency ratio of TRAM alone to the combined drug alone, Pl is the proportion of TRAM and P2 is the proportion of the combined drug in the total mixture (Sierralta et al., 1997). In the present study, fixed-ratio combinations were selected by choosing proportions that appeared to be near the line of additivity in which each compound was present in sub-analgesic doses, and testing them as described. The following drugs were used: tramadol hydrochloride, a gift of Grunenthal Chilena Ltd; clomipramine hydrochloride, a gift of Ciba-Geigy, Santiago, Chile; prazosin hydrochloride, xylamine hydrochloride and cyproheptadine hydrochloride, purchased from RBI, Natick, MA, USA. Results are presented as mean values +SE or as EDs0 values and 95% confidence intervals and were examined 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 (1987). Significance was accepted at the 0.05 level.

RESULTS Tramadol

antinociceptive

activity

The i.p. or i.t. administration of TRAM produced a dose-dependent antinociceptive activity meas-

345

0.1 Tramadol

1 (mg/kg)

FIG 1. Dose-response curves for effect of intraperitoneal (i.p.; 0) 0) administration of tramadol writhing test of mice.

10

100

the antinociceptive and intrathecal in the acetic

(i.t.; acid

ured by the acetic acid writhing test, with an EDso value of 7.83 + 1.15 and 1.82 f 0.57 mg/kg, respectively. The corresponding dose-response curves were not parallel (Fig. 1). Tramadol did not induce any visible change in motor activity.

Antinociceptive

interactions

of tramadol

PRA, CLON, XYL, CLOMI and CYPRO administered by the i.p. or the i.t. routes resulted in dose-dependent antinociceptive effects, and the corresponding EDjo values and potency ratios are shown in Table 1. The administration of these drugs did not induce behavioural, postural or motor changes. The EDso for the antinociceptive effect of the combinations of TRAM with these drugs in fixed-ratio mixtures appears in Table 2. The isobolographic analysis of these results show that simultaneous i.p. co-administration of the combinations produced antinociceptive effects greater than simple additivity and therefore the interaction was synergistic, since the ED,, point for each combination was significantly different from the corresponding theoretical EDSo, and they were located in the region of the isobologram that denotes superadditivity [Figs 2(a)-6(a)]. The analysis of the isobolograms obtained after the i.t. administration of TRAM plus PRA, CLON, XYL, CLOMI and CYPRO, revealed that the European

Journal

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(1998).

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G. PINARDI

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ETAL.

TABLE 1. ED,, values for antinociception and 95% confidence intervals for tramadol (TRAM), prazosin (PRA), clonidine (CLO), xylamine (XYL), clomipramine (CLOMI) and cyproheptadine (CYPRO) administered i.p. and i.t. in the writhing test of mice Drug

Ratio i.p/i.t.

E DsOmgkg (95% confidence interval) i.p.

TRAM PRAZ CLON (rig/kg) XYL CLOMI CYPRO

i.t.

7.83 (2.66-19.61) 1.28 (0.96-I .71) 414.31 (291.77-588.33) 14.63 (11.56-18.50) 1.09 (0.81-I .48) 0.74 (0.34-I .59)

1.82 (0.74-4.50) 0.13 (0.05-0.42) 36.53 (29.1 l-45.85) 0.49 (0.27-0.89) 0.77 (0.41-I .44) 0.064 (0.052-0.079)

4.3 9.8 11.3 29.8 1.4 11.6

TABLE 2. ED,, values and 95% confidence intervals for fixed-ratio combinations of tramadol (TRAM) and prazosin (PRA), clonidine (CLO), xylamine (XYL), clomipramine (CLOMI) and cyproheptadine (CYPRO) administered i.p. and i.t. in the writhing test of mice Combination

EDso mg/kg (95% confidence interval) Ratio”

i.p.

Ratio”

i.t.

TRAM:PRAZ

5.8:1

13.8:1

TRAM:CLON

175:O.Ol

1.66 (1.17-2.35) 0.13 (0.08-I .21) 5.33 (2.46-11.12) 0.89 (0.51-I .58) 0.26 (0.14-0.49)

0.63 (0.57-0.69) 0.54 (0.51-0.57) 0.88 (0.64-I .20) 0.81 (0.59-I .I 1) 0.41 (0.21-0.79)

TRAM:XYL

0.7:1

TRAM:CLOMI

7:l

TRAM:CYPRO

130:0.01

The combinations aratio tramadol:

DISCUSSION Tramadol administered either i.p. or i.t. displayed a dose-dependentantinociceptive activity measured by a chemical algesiometric test, but since Journal

of Pain

(1998),

1:0.2 1:0.3 400:0.01

are supra-additive or synergistic. other drug in combination, mg/kg.

antinociception induced by each combination was also superadditive in nature [Figs 2(b)-6(b)].

European

600:0.01

2

the i.p. and i.t. dose-responsecurves significantly deviate from parallelism, the effect must involve different mechanisms. Non-parallel curves suggest that the action is produced by activation of different pathways and not by activation of a common receptor (Goldstein et al., 1974).It has been demonstrated that TRAM is an antinociceptive, agent characterized by low affinity for opioid receptors, which is approximately lo-

ls0f30L0GRAP~ic

ANALYSIS

OF TRAMAD~L

347

INTERACTIONS

0.48

!

(a) 5

0.40

8 3

0.32

(a)

L

8 ’ 0.24 : z ‘5o B

0.16

I

0.08 I,

1

2 3 Tramadol

4 ED,,

5 (mg/‘kg,

6 i.p.1

7

8

0

1

2

3

Tramadol

4 ED,,

5

6

(mg/kg,

i.p.)

7

4o /

(b)

8

(‘b)

32 24 16 -

0.3

0.6 Tramadol

0.9 ED,,

1.2 (me/kg,

1.5 i.t.)

1.8

I

2.1

FIG. 2. lsobolograms for the co-administration of tramadol with prazosin. (a) Fixed-ratio combination (7.8:1) administered systemically (i.p.1. (b) Fixed-ratio combination (13.8:1) administered intrathecally (i.t.). Filled circles correspond to the theoretically calculated point with 95% confidence limits, and open circles correspond to the experimental point with 95% confidence limits.

fold less potent than codeine and 6000-fold less potent than morphine in the displacement of [3H] DAMGO from opioid binding sites in rat brain preparations (Raffa et al., 1992).This low range of affinity for opioid receptorsappearsinsufficient to explain its relatively high antinociceptive efficacy, and for this reason it seemednecessary to implicate other mechanisms that may contribute to the antinociceptive activity. The mechanism proposed is the inhibition of the neuronal uptake of noradrenaline and serotonin (Hennies et al., 1982;Driessen and Reimann, 1992;Raffa et al., 1992; Giusti et al., 1997). In addition, TRAM is systemically extensively metabolized to the M, active metabolite (O-desmethyl-tramadol), which binds to p-receptors with a higher affinity than the parent compond and may therefore contribute to the antinociceptive mechanism (Hennies et al., 1988; Friderichs and Becker,

S+ 0

0.6

0.9 Tramadol

1.2 1.5 ED,, (mg/kg, i.t.)

1.8

2.1

FIG. 3. lsobolograms for the co-administration of tramadol and clonidine. (a) Fixed-ratio combination (175:O.Ol) administered systemically (i.p.). B: Fixedratio combination (6OO:O.Ol) administered intrathecally (i.t.). Symbols as in Figure 2.

1991; Friderichs et al., 1992;Poulsen et al., 1996). Pain modulation by a large number of neurotransmitters has been implicated in the transmission of nociceptive messages(Wild and Raffa, 1996). Previous pharmacological characterization indicated that the inhibition of nociception by 01,- and a,-adrenoceptors was associatedwith the activation of the descending noradrenergic system (Howe et al., 1983;Wigdor and Wilcox, 1987);however, the antinociceptive action of CLON seems to be due to activation of cc,-adrenoceptorson primary afferent terminals at peripheral and spinal level (Eisenach et al., 1996).It has beenshown that qadrenergic agonists enhancethe analgesiainduced by intraspinal opioids, an interaction that occurs both pre- and post-synaptic to the primary afferent synapsein the spinal cord, and is clearly synergistic when both drugs are administered intrathecally (Ossipov et al., 1990~).In the present work, the coadministration of TRAM and CLON was shown European

Journal

of Pain

(I%@),

2

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G.

16

PINARDI

ETAL.

2 1.20 ; th. SC 6 1.00 -

(a)

5

0.80

-

g

0.60

-

2 '8

0.40

-

2 .g

0.20

-

(a)

5:

,o u 0

1

2 3 Tramadol

4 ED,,

5 (mg/kg,

6

7

8

0

1

i.p.)

2 3 Tramadol

4 ED,,

5 (mg/kg,

6

7

i.p.)

0.60

(b)

(b)

3

0

I, 0.3

I 0.6

Tramadol

I 0.9

I 1.2

ED,,

(mg/kg,

I

I 1.5

1

1.8

2.1

i.t.)

0.3

0.6

Tramadol

0.9

ED,,

1.2

1.5

(mgkg,

i.t.)

1.8

2.1

FIG. 4. lsobolograms for the co-administration of tramadol and xylamine. (a) Fixed-ratio combination (0.7:1) administered systemically (i.p.1. (b) Fixed-ratio combination (1:0.2) administered intrathecally (i.t.1. Symbols as in Figure 2.

FIG. 5. lsobolograms for the co-administration of tramadol and clomipramine. (a) Fixed-ratio combination (7:l) administered systemically (i.p.1. (b) Fixed-ratio combination (1:0.3) administered intrathecally (i.t.). Symbols as in Figure 2.

to be synergistic not only when administered intrathecally, but also when given systemically. This later finding probably indicates that the interaction can also occur at supraspinal levels, since CLON is rapidly and extensively absorbed into the CNS compartment. The antinociceptive activity displayed by PRAZ has been previously reported (Tjolsen et al., 1990;Kanui et al., 1993;Sierralta et al., 1996), indicating that a,-adrenoceptors are activated during the nociceptive transmission. The isoholographic analysis of the interaction between TRAM and PRAZ in the present work showed a synergistic effect in the acetic acid-induced writhings that resulted in enhanced antinociception. The antinociceptive action of PRAZ is not completely understood, but since PRAZ doesnot interfere with the liberation of substance P in the spinal cord (Ono et al., 1991), it is possible that it may block post-synaptic a,-adrenoceptors in the primary spinal afferent neurons involved in the nociceptive impulse

transmission. The synergy,evident both with systemic and intrathecal administration, could be attributed to modulation of the activity of the descending noradrenergic system that exerts a tonic inhibitory influence on the transmission of nociceptive information (Howe et al., 1983)and to an effect on primary neurons. XYL, a selective and irreversible inhibitor of noradrenaline uptake into central and peripheral noradrenergic neurones, enhanced antinociceptive activity of TRAM and the combination was clearly synergic when administered systemically or intrathecally. This finding is consistent with the proposed mechanism of action for (-) TRAM, i.e. blockade of noradrenaline neuronal uptake. On the other hand, CLOMI, an inhibitor of 5-HT uptake, was also synergistic when administered systemically or intrathecally in combination with TRAM, indicating that the effect of (+I TRAM is enhanced. This finding is in agreement with the suggestion that many

European

Journal

of fain

(1998).

2

iSOBOLOGRAPHIC

ANALYSIS

OF TRAMADOL

2 0.80 .A 2 i

2

349

INTERACTIONS

(a)

0.60

% 2 2 G it

0.40

t

0.20

4 1

2 3 Tramadol

4 ED,,

5 (mgkg,

6 i.p.)

7

8

(b)

0.3

0.6 Tramadol

0.9 ED,,

1.2 (mg/kg,

1.5 i.t.)

1.8

2’.1

FIG. 6. lsobolograms for the co-administration of tramadol and cyproheptadine. (a) Fixed-ratio combination (13O:O.Ol) administered systemically (i.p.). (b) Fixed-ratio combination (400:0.01) administered intrathecally (i.t.1. Symbols as in Figure 2.

antinociceptive drugs produce their action through a modulatory activation of serotonergic system receptors. Furthermore, CYPRO, an antagonist of 5-HT2 receptors, synergically potentiated the antinociceptive effect of TRAM, either systemically or intrathecally, indicating that different serotonin receptors are involved in antinociception. This role has been previously demonstrated for 5-HT2 (Eide and Hole, 1991) and 5-HT3 receptors (Pelissier et al., 1995; Hui et al., 1996) at spinal and supraspinal levels (Hamon et al., 1990). The pharmacological data presented in the present work on the antinociceptive activity of TRAM administered i.p. or i.t. suggest that TRAM activates receptors in the spinal cord and at central levels. In addition, it is confirmed that tramadol is not only an opioid agonist, but also acts through mono-aminergic mechanisms. Since no changes in muscle tone or behaviour were

observed with the administration of the drugs used, the synergiesobtained in the present work could be due to a complex modulation of the activity of the descendingnoradrenergic and serotonergic systemsthat have inhibitory influences on the transmission of nociceptive information, probably in addition to effects on the primary neuronsin the spinal cord. However, theseresults have been obtained using only a single animal pain model, but they are sufficiently positive to stimulate future researchwith other nociceptive models, e.g. thermal, mechanical, neuropathical, etc., and other analgesic drug combinations, in order to find possible negative interactions. On the other hand, the co-administration of analgesic drugs that produce superadditive effects constitute a significant new avenuefor the treatment of pain, considering that synergistic effects can result in a similar level of antinociception with considerablereductions in the dose of individual analgesicsand, consequently, with an important reduction in the incidence of side-effects. ACKNOWLEDGEMENTS The authors wish to thank J. Lopez and A. Correa for expert technical assistance. REFERENCES Budd K. Chronic pain. Challenge and response. Drugs 1994; 47 (Suppl.): 33-38. Driessen B, Reimann W. Interaction of the central analgesic, tramadol, with the uptake and release of 5-hydroxytryptamine in the rat brain in vitro. Br J Pharmacol 1992; 105: 1477151. Eide PK, Hole K. Different role of 5-HTIA and 5-HTz receptors in spinal cord in the control of nociceptive responsiveness. Neuropharmacology 1991; 30: 727-73 1. Eisenach JC, De Koch M, Klimscha W. cr,-adrenergic agonists for regional anesthesia. Anesthesiology 1996; 85: 655.674. Friderichs E, Becker R. Correlation of tramadol and M, serum level with antinociceptive activity in mice. NaunynSchmiedeberg’s Arch Pharmacol 1991; 343: R9. Friderichs E, Reimann W, Selve N. Contribution of both enantiomers to the antinociception of the centrally acting analgesic tramadol. Naunyn-Schmiedeberg’ Arch Pharmacol 1992; 346: R36. Giusti P, Buriani A, Cima L, Lipartiti M. Effect of acute and chronic tramadol on [3H]-5-HT uptake in rat cortical synaptosomes. Br J Pharmacol 1997; 122: 302-306. Goldstein A, Aronow L, Kalman SM. Drug Action: The European Journal

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