Adenosine Cyclophosphate

Effect of coadministration of caffeine and either adenosine agonists or cyclic nucleotides on ketorolac analgesia
Patricia Aguirre-Ban˜uelos, Gilberto Castan˜eda-Hern´andez, Francisco J. Lo´pez-Mun˜oz, Vinicio Granados-Soto )
Departamento de Farmacolog´ıa y Toxicolog´ıa, Centro de InÕestigacio´n y de Estudios AÕanzados del Instituto Polite´cnico Nacional, Apartado Postal 22026 14000 D.F. Mexico, Mexico

Abstract

Caffeine potentiation of ketorolac-induced antinociception in the pain-induced functional impairment model in rats was assessed. Caffeine alone was ineffective, but increased the effect of ketorolac without affecting its pharmacokinetics. Intra-articular administration of adenosine and N 6-cyclohexyladenosine ŽCHA, an adenosine A 1 receptor agonist., but not 2-p-Ž2-carboxyethyl.phenethylamino-5X-N- ethylcarboxamidoadenosine hydrochloride ŽCGS-21680, an adenosine A 2A receptor agonist., significantly increased ketorolac antinoci- ception. This effect was not local, as contralateral administration was also effective. Ipsilateral and contralateral administration of adenosine and CHA also increased antinociception by ketorolac–caffeine. Intra-articular 8-Bromo-adenosine cyclic 3X,5X-hydrogen phosphate sodium or 8-Bromo-guanosine-3X,5X-cyclophosphate sodium ŽcGMP.

given ipsilaterally or contralaterally did not affect

ketorolac-induced antinociception. Nevertheless, ipsilateral, but not contralateral, administration of 8-Br-cGMP significantly increased antinociception by ketorolac–caffeine, suggesting a local effect. The results suggest that caffeine potentiation of ketorolac antinociception is mediated, at least partially, by a local increase in cGMP and rule out the participation of adenosine receptor blockade. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Ketorolac; Caffeine; Potentiation; Adenosine A 1 receptor; cGMP

1.Introduction

There are a number of reports that caffeine can potenti- ate the antinociceptive effects of non-steroidal anti-in- flammatory drugs ŽNSAIDs. in animal models ŽSiegers, duced evidence that caffeine can indeed improve analgesic efficacy, but only in certain pain states and dose ratios ŽLaska et al., 1984; Forbes et al., 1991; Sawynok and Yaksh, 1993.. Despite the fact that potentiation of the antinociceptive effect of NSAIDs by caffeine has been 1973; Vinegar et al., 1976; Seegers et al., 1980; Granados-Soto et al., 1993.. In humans, caffeine has been extensively used as an analgesic adjuvant ŽLaska et al., 1984; Forbes et al., 1991; reviewed Sawynok and Yaksh, 1993.. However, the ability of caffeine to increase the analgesic effect of NSAIDs has been questioned, since there are reports that caffeine is ineffective to increase the attributed to the inhibition of adenosine receptors ŽSawynok and Yaksh, 1993., the mechanism of such potentiation is not well understood. Therefore, we decided to determine whether the mechanism of this synergy is pharmacokinetic or pharmacodynamic. In addition, we studied the potentia- tion by caffeine of the antinociceptive activity of ketorolac in the presence and absence of cyclic nucleotides Ž8-antinociceptive effect of NSAIDs ŽCass and Frederik, 1962;
Moertel et al., 1974.. In contrast, in the last decade an increasing number of controlled clinical trials have pro-Bromo-adenosine cyclic 3X,5X-hydrogen phosphate monosodium, 8-Br-cAMP and 8-Bromo-guanosine-3X,5X- cyclophosphate sodium, 8-Br-cGMP. and adenosine ago- nists Žadenosine, N 6-cyclohexyladenosine ŽCHA. and 2-p- Ž2-carboxyethyl.phenethylamino- 5X-N -ethylcarboxamido- adenosine hydrochloride ŽCGS-21680.. ŽFredholm et al., 1996. by way of assessing the participation of peripheral cyclic nucleotides and adenosine receptors activation, re- spectively, in this action.

2.Material and methods

2.1.Animals

Female Wistar rats Žweight range, 180–220 g. from our own breeding facilities wCrl:ŽWI.BRx were used in this study. Twelve hours before the initiation of experiments, food was withheld, but the animals had free access to drinking water. Rats to be used in the pharmacokinetic study were lightly anaesthetized with ethyl ether. Then,of discomfort, such as licking, biting, shaking, elevating, vocalization.
As a result of uric acid injection, the rats developed a progressive dysfunction of the injured limb. This was recorded as a diminished time of contact between the right hind limb and the cylinder. Data are expressed as the functionality index, i.e., the time of contact of the injected limb divided by the time of contact of the control left limb multiplied by 100. After 2 h, the injected limb made no contact with the cylinder, the rats received the drugs and recordings were carried out during the next 4 h. Recovery of the functionality index was considered as the expression of the antinociceptive effect.

2.4. Ketorolac determination

polyethylene catheters Ža combination of a PE-10 and PE-50 was used; i.d. 0.28 mm, o.d. 0.61 mm and i.d 0.58 mm, o.d. 0.96 mm, respectively, Clay Adams, Parsippany, NJ, USA. were surgically implanted into the caudal artery for the collection of blood samples. All experiments fol- lowed the Guidelines on Ethical Standards for Investiga- tion of Experimental Pain in Animals ŽZimmermann, 1983.. Additionally, the study was approved by the local Animal Care Committee.

2.2. Drugs

Racemic ketorolac tromethamine was obtained from Roche-Syntex ŽMexico City.. Caffeine, adenosine, 8-Br- cAMP, 8-Br-cGMP and uric acid were purchased from

Blood concentrations of ketorolac were determined by a high performance liquid chromatographic ŽHPLC. method previously described ŽFlores-Murrieta et al., 1994..

2.5. Study design

In the first experimental series, six groups of at least six rats each were used to test the potentiation by caffeine of the antinociceptive effect of ketorolac. Once the function- ality index was zero, the animals received vehicle Žcarboxymethyl cellulose., 1.8 mgrkg of ketorolac tromethamine, 32 mgrkg of caffeine or the combination of Žadenosine A 1 and A 2A-selective receptor agonists, respec- tively. ŽFredholm et al., 1996. were purchased from Re- search Biochemical International ŽNatick, MA, USA.. Acetonitrile was chromatographic grade ŽMerck, Darmstadt,Germany.. Deionized water was obtained through a Milli-Q system ŽContinental Water Systems, El Paso, TX, USA.. Other reagents used in this study were of analytical grade.

2.3. Measurement of antinociceptiÕe actiÕity

Antinociception was assessed with the pain-induced functional impairment model in the rat, as described previously ŽLo´pez-Mun˜oz et al., 1993.. Nociception was induced by the intra-articular injection of 50 ml of 30% uric acid suspension in mineral oil into the right hind knee and an electrode was attached to each hind limb behind the plantar pads. At selected times, the rats were required to walk on a cylinder of 30-cm diameter rotating at 4 rpm for 2-min periods. The variable measured in this model was the time of contact of each electrode with the cylinder floor. When the electrode placed on the animal’s paw made contact with the cylinder floor, a circuit was closed and the time that the circuit remained closed was recorded. The animals were allowed to rest between recording peri- ods. During resting periods the rats did not show any sign

Fig. 1. Area under functionality index against time curve ŽAUCE ., which represents a global antinociceptive effect, observed in rats with pain-in- duced functional impairment by intra-articular injection of 30% uric acid in the right hind knee. Rats received ketorolac alone Ž1.8 mgrkg. or combined with 10, 14, 18 or 32 mgrkg caffeine. Data are expressed as means”S.E.M. of six determinations. a Significantly different from the ketorolac–caffeine combination alone Ž P -0.05., as determined by anal- ysis of variance followed by Dunnett’s test.

Fig. 2. Time course of the whole blood ketorolac levels observed in rats with pain-induced functional impairment by intra-articular injection of 30% uric acid in the right hind limb. Animals received 1.8 mgrkg of ketorolac tromethamine Žclear circles. alone or combined with 32 mgrkg of caffeine Ždark circles.. Data are expressed as means”S.E.M. of six determinations.

ketorolac tromethamine Ž1.8 mgrkg. with 10, 14, 18 or 32 mgrkg of caffeine dissolved in 0.5% carboxymethyl cellu- lose by gavage. Functionality index was evaluated at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 h after drug administration. Ketorolac and caffeine doses were selected based on previ- ous studies ŽGranados-Soto et al., 1993, 1995..
In the second experimental series, we examined whether the potentiation of ketorolac-induced antinociception by caffeine had a pharmacokinetic mechanism. For this pur- pose, 1.8 mgrkg of ketorolac tromethamine alone or com- bined with 32 mgrkg of caffeine was given. Whole blood 8-Br-cGMP, CHA or CGS-21680 and simultaneously a dose of ketorolac tromethamine Ž1.8 mgrkg. or the combi- nation of ketorolac–caffeine Ž1.8–14 mgrkg p.o…
2.6. Data analysis and statistics

Maximal blood concentrations ŽCmax . and time to reach the maximal blood concentration Žtmax . were determined directly from the individual blood concentration against time curves. The area under the ketorolac blood concentra samples

Ž100 ml.

were obtained, through the cannula

inserted into the caudal artery, at 0, 15, 30 and 45 min and
at 1, 1.5, 2, 2.5, 3, 3.5 and 4 h after drug administration. Then, blood samples were frozen and stored at y708C until analyzed. In the third experimental series, once func- tionality index had reached zero, the animals received either ipsilaterally or contralaterally an intra-articular injec- tion of increasing doses of saline, adenosine, 8-Br-cAMP,

Table 1
Values for pharmacokinetic and pharmacodynamic parameters of ketoro- lac obtained after oral administration of ketorolac tromethamine alone Ž1.8 mgrkg. or combined with 32 mgrkg of caffeine. Data are expressed as means”S.E.M. of six determinations
Parameter Ketorolac Ketorolac–caffeine
Cmax Žmgrml.
tmax Žh. 2.38″0.26
1.25″0.43 2.72″0.28
0.75″0.43
AUC Žmg hrml. 6.70″0.58 7.50″0.81

Eobs Ž%.
tEmax Žh.
AUCE Ž% h.

66.0″6.72 85.4″4.86a
1.58″0.20 1.16″0.10
145.9″11.1 213.1″21.9a

Fig. 3. Effect of ipsilateral or contralateral intra-articular administration of adenosine, ADO ŽA., CHA Žadenosine A1 receptor agonist. ŽB. and CGS-21680 Žadenosine A 2A receptor agonist. ŽC. on ketorolac Ž1.8 mg
rkg. antinociception. Data are expressed as the AUCE for six animals”Analysis of variance followed by Dunnett’s test was used to test differences in the potentiation studies. Compar- ison between pharmacokinetic parameter values observed with ketorolac alone and combined with caffeine were performed by the Student’s t-test for unpaired data.

3.Results

3.1.Potentiation of ketorolac antinociception by caffeine
Fig. 4. Effect of ipsilateral or contralateral adenosine, ADO ŽA., CHA Žadenosine A1 receptor agonist. ŽB. and CGS-21680 Žadenosine A 2A receptor agonist. ŽC. on ketorolac–caffeine Ž1.8–14 mgrkg. antinocicep reached a peak of about 65% and then decayed gradually. Caffeine alone was not able to produce any significant antinociceptive action, but when it was coadministered with ketorolac, it induced a significant dose-dependent increase in the antinociceptive effect. The antinociceptive effect of the combination of either 10 or 14 mgrkg caffeine and 1.8 mgrkg of ketorolac was not different from that of ketorolac alone Ž P ) 0.05.. However, subse- quent doses of caffeine Ž18 and 32 mgrkg. produced a clear potentiation of ketorolacs antinociceptive activity Ž P – 0.05. ŽFig. 1tion. Data are expressed as the AUCE for six animals”S.E.M. a indicates a significant difference against ketorolac–caffeine combination alone Ž P -0.05., as determined by analysis of variance followed by Dunnett’s test.

Curves were made for functionality index against time and the maximal antinociceptive effect Ž Eobs . and time to were directly determined from these plots. The area under the functionality index against time curve ŽAUCE ., considered as an expression of the overall antinociceptive activity during the 4-h observation period, was estimated by the trapezoidal rule ŽLo´pez-Mun˜oz et al., 1993..

Fig. 5. Effect of ipsilateral or contralateral intra-articular administration of 8-Br-cAMP ŽA. and 8-Br-cGMP ŽB. on the ketorolac antinociception. Data are expressed as the AUCE for six animals”S.E.M.

3.2.Ketorolac–caffeine pharmacokinetic interaction

adenosine A 1 receptor agonist, CHA Ž50 nmolrknee., but

Since a clear potentiation was observed with 1.8–32 mgrkg ketorolac–caffeine, we decided to study this com- bination in comparison with 1.8 mgrkg ketorolac alone in order to study a possible pharmacokinetic interaction. Ke- torolac blood concentrations were similar in the presence and absence of caffeine ŽFig. 2., despite the fact that the association, ketorolac– caffeine, produced a higher antinociceptive effect than ketorolac alone ŽFig. 1.. Caf- feine did not produce any significant modification of ke- torolac pharmacokinetics, although the values of pharma- codynamic parameters Eobs and AUC were significantly Ž P – 0.001. increased ŽTable 1.. Caffeine pharmacokinet- ics were not examined as this drug, by itself, failed to produce any significant antinociception.

3.3.Adenosine agonists

Adenosine receptor agonists Žadenosine, CHA and CGS-21680. were not able to produce any effect when administered to either normal or injured animals Ždata not shown.. Either the ipsilateral or contralateral intra-articular administration of adenosine Ž50 nmolrknee. and the

Fig. 6. Effect of ipsilateral intra-articular administration of 8-Br-cAMP ŽA. and ipsilateral or contralateral 8-Br-cGMP ŽB. on the ketorolac–caf- feine Ž1.8–14 mgrkg. antinociception. Data are expressed as the AUCE for six animals”S.E.M. a indicates significant difference compared to the ketorolac–caffeine combination alone Ž P -0.05., as determined by analysis of variance followed by Dunnett’s test.

not the adenosine A 2A receptor agonist, CGS-21680, si- multaneously with the oral administration of ketorolac significantly increased antinociception Ž P – 0.05. ŽFig. 3.. Ipsilateral or contralateral adenosine, at the same dose level, was also able to significantly increase the antinoci- ceptive effect of ketorolac–caffeine. Similar results were observed with CHA, although the dose of this adenosine
A 1 agonist required to potentiate the effect of ketorolac– caffeine was 100 times lower than that needed to increase the effect of ketorolac alone Ž0.5 versus 50 nmolrknee. ŽFig. 4..

3.4.Cyclic nucleotides

8-Br-cAMP or 8-Br-cGMP were not able to produce any significant effect when given to either normal or injured rats Ždata not shown.. Furthermore, the effect of ketorolac alone was not altered by intra-articular adminis- tration of increasing doses of 8-Br-cAMP or 8-Br-cGMP given either ipsilaterally or contralaterally simultaneously with oral ketorolac ŽFig. 5.. In contrast, ipsilateral, but not contralateral, administration of 8-Br-cGMP significantly increased Ž P – 0.05. the antinociceptive effect of the ke- torolac–caffeine combination ŽFig. 6.. 8-Br-cAMP failed to increase the antinociceptive effect of ketorolac–caffeine.

4.Discussion

4.1.Potentiation of ketorolac antinociception by caffeine

Oral administration of caffeine did not produce antinociception in our model of hyperalgesia. However, it increased the antinociceptive effect of ketorolac in a dose- related manner. The caffeine dose that produced the best potentiation Ž18 or 32 mgrkg. in this study was similar to that used in combination with paracetamol ŽGranados-Soto et al., 1993. and tolmetin ŽFlores-Acevedo et al., 1995.. These data confirm previous observations on the ability of caffeine to potentiate the effect of NSAIDs in experimental pain models ŽVinegar et al., 1976; Seegers et al., 1980; Gayawali et al., 1991; Granados-Soto et al., 1993;
Castan˜eda-Hern´andez et al., 1994; Flores-Acevedo et al., 1995. as well as in clinical situations ŽLaska et al., 1984..
Caffeine has been demonstrated to produce antinocicep- tion in threshold ŽPerson et al., 1985; Malec and Michal- ska, 1988. and hyperalgesic tests ŽSiegers, 1973; Seegers et al., 1980; Sawynok et al., 1995.. In contrast, there are a number of studies which have not detected antinociception with caffeine in both threshold and hyperalgesic studies ŽFialip et al., 1989; Granados-Soto et al., 1993; Castan˜eda-Hern´andez et al., 1994; this study.. Contradic- tory results of these studies are likely due to the use of different experimental models to detect antinociception.

4.2.Ketorolac–caffeine pharmacokinetic interaction
It has been suggested that the potentiation by caffeine could be due to an increase of NSAID bioavailability through an increase its absorption or impairment of its and ketorolac–CHA, but not that produced by ketorolac- CGS-21680. Malmberg and Yaksh Ž1993. have reported an additive synergism of the combination of ketorolac and N 6-ŽL-2-phenyilisoptopyl.-adenosine ŽL-PIA., an adenosine A 1 receptor agonist. As in this study, the addition of elimination ŽSawynok and Yaksh, 1993.. Our results, however, are not consistent with a pharmacokinetic interaction. When caffeine augmented the antinociceptive effect of ketorolac, the blood levels of the NSAID were similar to those observed in the absence of the xanthine, without any significant alteration of pharmacokinetic parameters. Al- though it has been reported that caffeine can modify gastric acidity, as well as gastric and hepatic blood flows ŽDebas et al., 1971; Beubler and Lambech, 1976; Onrot et al., 1986., these actions appear to have no relevance for the bioavailability of ketorolac. Our data are consistent with those previously reported for the combination of caffeine with paracetamol ŽGranados-Soto et al., 1993., aspirin ŽVinegar et al., 1976; Collins et al., 1979;
Castan˜eda-Hern´andez et al., 1994. and tolmetin ŽFlores- Acevedo et al., 1995., but disagree with those of Siegers Ž1973. and Seegers et al. Ž1980..
4.3.Adenosine agonists
Under our conditions, the adenosine agonists were not able to produce nociception or antinociception, but they increased the activity of ketorolac or the ketorolac–caf- feine combination. The intra-articular administration of adenosine or the adenosine A 1 receptor agonist, CHA, but caffeine, at a dose that antagonized L-PIA alone, failed to affect the combination ketorolac-L-PIA in the second phase of the formalin test. Therefore, our results are consistent with those of Malmberg and Yaksh Ž1993.. However, our data are in contrast with other reports ŽKarlsten et al., 1992; Poon and Sawynok, 1998. in which theophylline or caffeine, non-specific antagonists of adenosine receptors, were able to block the antinociception produced by local or spinal administration of adenosine receptor agonists. Our results suggest that the effects of caffeine are not due to the blockade of the adenosine receptors, but that other effects of the xanthine are involved. However, it is also possible that there is a certain degree of adenosine antago- nism, but that caffeine actions in other compartments predominate in such a way that the global result is a potentiation of the antinociceptive effect. It is not probable that caffeine produces antinociception or potentiation of NSAID-induced antinociception by a blockade of adeno- sine receptors, as it has been reported that spinal and supraspinal administration of adenosine analogs produces antinociception by activation of adenosine A 1 receptors ŽPost, 1984; Karlsten et al., 1990.. Therefore, other effects of caffeine must be responsible for such antinociception or potentiation. The potentiation of the endogenous choliner not the adenosine A 2A receptor agonist, CGS-21680, significantly increased antinociception by ketorolac. This ef- fect was not due to the local effect of the agonists, as the contralateral administration of adenosine and CHA also significantly increased antinociception. These data suggest that adenosine is not producing a nociceptive effect, but facilitates the effect of ketorolac by a spinal or supraspinal action. Our results are supported by those studies of in vivo models of nociception which have suggested that the adenosine A1 receptor plays an important role in spinal antinociception by inhibiting sensory transmission related
to nociceptive information at the spinal level ŽReeve and Dickenson, 1995; Nakamura et al., 1997; Poon and Sawynok, 1998.. In our model, we were not able to observe any antinociceptive effect with the adenosine re- ceptor agonists. This discrepancy with previous studies could be due to the intensity of the nociceptive stimuli used. Recently, it has been reported that the antinocicep- tion produced by adenosine is intensity-dependent ŽSawynok et al., 1998..
In addition to their effect on the antinociception pro- duced by ketorolac, adenosine and CHA, but not CGS- 21680, also increased the effect of the ketorolac–caffeine combination. Caffeine was not able to block the potentia- tion of the effect of adenosine agonists on ketorolac-in- duced antinociception. In fact, the addition of caffeine significantly increased the effect of ketorolac–adenosine the activation of central noradrenergic pathways which regulate nociceptive thresholds has been suggested to play an important role in antinociception by caffeine in the several pain tests ŽSawynok et al., 1995.. Other effects, such as inhibition of phosphodiesterases ŽChoi et al., 1988., nitric oxide release ŽLo´pez-Mun˜oz et al., 1996. and stimu- lation of the central nervous system could also be con- tributing to the adjuvant effect of caffeine.
4.4.Cyclic nucleotides
We have previously reported that local administration of N G-nitro-L-arginine methyl ester, a nitric oxide synthe- sis inhibitor, is able to block the potentiation by caffeine of
ketorolac-induced antinociception ŽLo´pez-Mun˜oz et al., 1996.. These data suggest a participation of the L-
arginine-nitric oxide-cGMP pathway in antinocieption. In the present study we found that intra-articular administra- tion of 8-Br-cAMP or 8-Br-cGMP, by themselves, did not produce any effect. Furthermore, these compounds were not able to produce any effect on the antinociception of oral ketorolac when given either ipsilaterally or contralat- erally. In contrast, intra-articular administration of 8-Br- cGMP significantly increased Ž P – 0.05. the effect pro- duced by the ketorolac–caffeine combination when in- jected ipsilaterally, but not contralaterally. These results suggest a local action of 8-Br-cGMP. Conversely, 8-Br- cAMP did not produce any significant change of the ketorolac–caffeine effect. These results can be interpreted in the light of the results of Ferreira and Nakamura Ž1979. and Taiwo and Levine Ž1990., who reported an involve- ment of cGMP in the production of antinociception. The fact that 8-Br-cGMP, by itself, failed to induced any significant effect and also failed to potentiate the effect of ketorolac can be explained by a rapid degradation of the nucleotide. As caffeine is a nonspecific inhibitor of phos- phodiesterases ŽChoi et al., 1988. it is likely that 8-Br- cGMP administration in the presence of caffeine was effective as its degradation was prevented. The available data suggest that caffeine could be producing the potentia- tion of NSAIDs by increasing cGMP or inhibiting its degradation at the site of inflammation. This suggestion is supported by the report of Hatano et al. Ž1995. that caffeine increases cGMP in two ways, by increasing nitric oxide and subsequently cGMP, or by inhibiting cGMP degradation by phosphodiesterase inhibition. In addition, it has been shown recently that nitric oxide can enhance antinociception in several models of hyperalgesia ŽGrana- dos-Soto et al., 1997; Nozaki-Taguchi and Yamamoto, 1998..
In summary the data provided here indicate that potentiation by caffeine of the antinociceptive effect of ketoro- lac is not due to a pharmacokinetic interaction, but to a pharmacodynamic mechanism. The actions of caffeine ap- pear to be complex, involving several mechanisms. Our results suggest that, in this model, adenosine does not have any pronociceptive or antinociceptive effect by itself, but is able to facilitate ketorolac-induced antinociception by activation of the adenosine A 1 receptor, probably at the central level. The potentiation of ketorolac by caffeine does not appear to result from the antagonism of pronoci- ceptive adenosine actions. Our data suggest a peripheral effect of caffeine, involving local cGMP increase as a mechanism of potentiation of NSAID-induced antinocicep- tion. Additionally, there is evidence for a central antinoci- ceptive action of caffeine which is also likely to be in- volved in the potentiation of NSAID effects by this xan- thine ŽSawynok et al., 1995; Ghelardini et al., 1997..

Acknowledgements

The authors wish to thank Mr. A. Huerta and L. Oliva for technical assistance. Patricia Aguirre-Ban˜uelos is a CONACYT fellow. Work supported by a CONACYT Grant 0250-P-M.

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