Gen. Pharmac. Vol. 24, No. 3, pp. 693-698, 1993 0306-3623/93 $6.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1993 Pergamon Press Ltd

KETOROLAC TROMETHAMINE: AN EXPERIMENTAL STUDY OF ITS ANALGESIC EFFECTS IN THE RAT

DIEGO BUSTAMANTE and CARLOS PAEILE* Department of Pharmacology, Faculty of Medicine, University of Chile, Santiago, Chile

(Received 2 November 1992)

Abstract--l. The formalin test and R-III nociceptive electromyographic reflex were used to determine the origin of analgesia induced by ketorolac tromethamine (KT) in rats.

2. The effects of KT and morphine were compared after i.v. administration. 3. Mepacrine and indomethacin were associated to KT to determine if prostaglandins are involved in

the central action of KT. 4. In both tests KT had a poor analgesic effect without dose-response relationships. 5. A central component is involved in the analgesia produced by KT, but neither prostaglandins nor

opioid receptors seem to mediate this effect.

INTRODUCTION

Ketorolac tromethamine (KT) is a non-steroidal anti- inflammatory drug (NSAID) for oral and parenteral use (Buckley and Brodgen, 1990). Clinical studies tend to show that its efficacy as an analgesic is greater than other NSAIDs, and is similar to morphine (Yee et al., 1984, 1986; Brown et al., 1988; Goodman, 1991); however there are only a few experimental reports related to the study of its analgesic effects (Rooks et al., 1982, 1985; Domer, 1990).

Because of its anti-inflammatory, antipyretic, analgesic and antiplatelet properties, it has been proposed that the mechanism of action of K T may be related to the inhibit ion of prostaglandin biosynthesis at the cyclooxygenase level (Rooks et al., 1982). Nevertheless, the relative potency described in a number of clinical studies suggests the possibility that other mechanisms might also be involved in the antinociceptive action of this drug.

We are interested in the mechanisms involved in the central effects of NSAIDs, and in previous studies we have demonstrated that indoprofene and lysine clonixinate are central components in their analgesic effects (Paeile et al., 1989; Bustamante et al., 1989), as well as that mefenamic acid modifies the electro- physiological activity of some cerebral structures (Pelissier et al., 1984).

Based on clinical data suggesting that K T and morphine would display a similar analgesic potency, this experimental study was conducted to compare the analgesic potency of K T with that of morphine in

*To whom all correspondence should be addressed at: P.O. Box 70.000, Santiago 7, Chile.

693

two experimental algesiometric tests, and to investigate the possibility that a central antinociceptive effect might play a role in the mechanism of action of KT, as has been demonstrated for salicylates, ketoprofene and others drugs (Dubas and Parker, 1971; Willer and Harrewyn, 1987). Considering that an important anti-inflamatory action for K T has been demon- strated, it was interesting to find out whether inhibi- tion of prostaglandins modified its analgesic effects, and for this purpose the interaction with mepacfine and indomethacine was studied.

MATERIAL AND METHODS Animals

Adult Wistar rats, weighing 220-270 g were used Jn both tests. They were maintained in a climate-controlled room (21 _+ I°C) with a natural dark-light cycle and free access to food and water prior to the experiments, which took place during the first hours of the light phase. Each animal was used only once and was sacrificed with an overdose of anesthetic immediately after the experiment.

Tests o f antinociception

Formalin test. This algesiometric assay was performed following the rating scale for rats described by Dubuisson and Dennis (1977), exposing the animals individually to the observation chamber (30 x 30 x 30 cm) for 1 hr prior to testing.

Data recording was initiated immediately after the 50 #1 injection of 5% formaldehyde solution subcutaneously into the dorsal hind paw. The pain responses were evaluated assigning numerical values from 0 to 3, which reflected the perceived pain intensity. Briefly, the rating scale is as follows:

0: Both forepaws are placed on the floor, and weight is evenly distributed; during locomotion, there is no discernible favoring of the injected paw;

1: the injected paw rests tightly on the floor or in another part of the animal's body or no weight is placed upon it; during locomotion there is no obvious limp;

PAIN INYIr.NsITY RATING 3.0~

2.G

2.0

1.5

1 . 0 . . . . .

O.G

0.0

694 Dm3o BUSTAMANTE and CARLOS PAEILE

0 3 6 9 12 13 18 21 24 27 30 n ~ t .

Fig. 1. Temporal evolution of pain intensity rating in rats subjected to the formalin test and treated with saline (0.9% NaCI) (11), morphine 10 (~) , KT 80 (A), mepacrine 20 ( ~ ) and mepacrine 20 plus KT 80 0-3). Doses i.p. are expressed in mg/kg. Data represent the mean, for a number of experiments n/>4.

SEM bars are omitted for clarity.

2: the injected paw is elevated and not in contact with any surface;

3: the injected paw is licked, bitten or shaken, while the uninjected paw is not.

Observation of each animal took place for a 30 min period after which the ratings were transformed to a numerical value using the formulae proposed by the authors. In order to analyze the temporal course of the analgesia during the whole period, partial scores were calculated each 3 min (Fig. 1).

Drugs tested for analgesic properties or saline (0.9% NaC1) were injected intraperitoneally 30 rain before the noxious stimulus, using a volume of 1 ml/kg body weight.

Bicepsfemoris nociceptive reflex. This method is based on the observation of Wilier (1977), who demonstrated a close relationship between the amplitude of the R-III bioelectrical signal recorded at biceps femoris with the painful sensation perceived by men voluntarily subjected to the nociceptive electrical stimulation of the receptive field of the sural n e r v e .

In animals this method has been validated by Falinower et al. (1991). The electrical noxious stimuli were applied to anesthetized rats, to the right sural nerve receptive field (toes 4 and 5) and originated in a Grass S-44 stimulator adjusted to give rectangular pulses of 2 msec duration and 30 V intensity every I0 sec.

Stimulation of C-fibers evoked an R-III electromyographic (EMG) response that was recorded using 2 needle electrodes inserted through the skin over the right biceps femoris muscle. EMG responses were amplified in a Grass RPS 107 bioamplifier, exhibited in a Tektronic 2205 oscilloscope and also digitized with an analog-digital converter for further process by an IBM-PC. Specially designed software allowed exhibition the EMG signal (Fig. 2) and of the average mean of the spikes observed between 150 and 450 msec after the stimulus (R-III response evoked by C-fiber stimulation), which was printed as a histogram (Fig. 3).

Signals and histograms reflecting the temporal course of analgesia were used to analyze the results. Table 2 shows the mean value calculated for the average amplitude of 20 signals (approx. 3 min) prior to and post-drug administra- tion. The basal electrical muscular noise, which was never greater than 10% of the reflex signal amplitude was deducted from the average amplitude. Saline did not modify signal amplitude.

Drugs were injected i.v. in a volume of I ml/kg body weight through a heparinized cannula previously inserted in the contralateral femoral vein of urethane (1 g/kg) anesthetized rats.

Statistical analysis The data were examined by analysis of variance (ANOVA

one-way) for multiple comparisons with a single control

I V

O V

- I

(a) I S .h l l

I V

)_V

- l i

(b ) (c) S=t*l I SePal }

"~" ' " ' i ' " ' " f '":. ............. ~ " - ' ! ' " ' " ' i ' " ' " ' i " [ 1 V ii I ) ; ;

. . . . . . . .

: O V . . . . T~r,r,,T ~- ~ - T - - : --- ,.......!.......!.......?...... .........!...... !...-.!.....!.......!........!.....!.....! .......

~'"'-'--~"'"";'""'~ .. . . .'"'"'~'"'"';'"''~""--.. . "~--i'--! "!-"" i ""'i"" ! - i ....... ...... -~-..-.--::....-..,........-,~.-..--i..-...i...--..i . . . . . . . . . . . i :,. :~ - - - . . i - -~- : . - :~ ..:, i ....

: : : : : : : : : : : : : . : : .

I 100 msec

Fig. 2. Representative muscular R-III reflex signals, recorded at the biceps femoris of the rat after nociceptive stimulation of the ipsilateral toe (a) under control conditions, (b) after treatment with 80 mg/kg

KT and (c) after treatment with 10 mg/kg morphine sulphate.

Ketorolac experimental analgesia

- I H i s t o ~ r a * a [

695

T T T T T T ',.,. 5 t 20 4 0 80 t 20 M N I K T '

Fig. 3. Histogram showing the temporal evolution (50 min) of the amplitude of the R-III reflex nociceptive signals in biceps femoris of rats treated with 5 mg/kg morphine sulphate (M), 1 mg/kg naloxone (N) and

20-120 mg/kg KT. See Methods for details.

group, but when the analysis was restricted to two means, Student's t-test (two tails) was used. Level of significance was set at 5% (P<0.05). Results are expressed as mean + SEM.

Drugs

Formaldehyde 37% solution, sodium chloride and morph- ine hydrochloride were obtained from Merck Chemical Co., Darmstadt, Germany; naloxone was purchased from Endo Labs Inc., New York, U.S.A; indomethacin, urethane, mepacrine (quinacrine dihydrochloride) and heparin from Sigma Chemical Co., St Louis, Mo, U.S.A. and ketorolac tromethamine was obtained from a commercially available source.

RESULTS

Formalin test

The whole intensity ratings up to 30 min, calculated according to Dubuisson and Dennis (1977), are shown in Table I.

The control group, treated with saline, showed the highest algesiometric score, and the groups treated with KT 40, 80, 120 or 200 mg/kg, showed a slight but statistically significant analgesia (P < 0.05), while KT 20 mg/kg did not. However, it is noticeable that no changes in analgesic effect were seen when doses were increased, indicating the lack of dose-response relationships.

Indomethacin and mepacrine (20mg/kg), both potent inhibitors of prostaglandin synthesis, also showed a slightly significant analgesia (P<0.05) when administered 1 hr before testing. The effect was increased with 24 hr of drug action (Table 1).

The association of KT (80 mg/kg) with indo- methacin or mepacrine failed to modify the individual algesiometric scores and only the interaction with mepacrine administered 1 hr before, increased KT analgesia (P <0.05).

Temporal evolution of partial pain intensity ratings each 3 min, used to obtain information about the phases in the animal's response to the nociceptive stimulus, showed that the maximal analgesic efficacy

Table 1. Algesiometric scores of control and treated groups in the formalin test in the rat*

Dose Algesiometric P vs Drug (mg/kg) score control

Saline 2,279 _+ 0.032 (22) - -

Morphine 2 2,135 -+ 0.069 (6) ns 5 0,890 _+ 0.090 (6) < 0.0001

l0 0.800_+0.140(6) < 0.0001

Ketorolac 20 2.368 _+ 0.129 (4) ns 40 1.839 -+ 0.055 (4) <0.05 80 1.899 _+ 0.027 (6) <0.05

120 2.042 _+ 0.034 (4) <0.05 200 1.911 +0 .014(4) <0.05

Indomethacin 20 (I hr) 2.027 -+ 0.047 (13) <0.05 (24 hr) 1.808 -+ 0.190 (4) < 0.05

Mepacrine 20 (1 hr) 2.120 -+ 0.030 (4) <0.05 (24 hr) 1.778 -+ 0.036 (4) < 0.001

Associations: Indomethacin 20 (1 hr) + ketorolac 80 lndomethacin 20 (24 hr) + ketorolac 80 Mepacrine 20 (I hr) + ketorolac 80 Mepacrine 20 (24 hr) + ketorolac 80

2.061 +_ 0.053 (6) 2.000 _+ 0.063 (4) 1.757 + 0.072 (4)'t" 1.882 _+ 0.066 (6)

*Data are expressed as the mean ±SEM, for the number of animals (n) in parentheses.

~'P < 0.05 vs KT 80 mg/kg and mepacrine 20 mg/kg (1 hr) (Students t-test).

696 DiEoo BUSTAMANTE and CARLOS PAEILE

of KT was obtained between 10 and 20 min after formalin injection. Mepacrine (20 mg/kg) administered 1 hr prior to injection was unable to modify the control values, but when administered 24 hr before, produced an analgesia that was very significant compared to controls around 15 min after the nociceptive stimuli.

The temporal evolution of the algesiometric scores did not differ significantly in control rats and in rats treated with both KT (80 mg/kg) and indomethacin (20 mg/kg). However, when KT was associated with mepacrine (1 or 24 hr prior to the assay) a significant difference to the control group, but not with KT alone, was observed (Fig. 1).

Morphine 2mg/kg, did not show a significant analgesic activity in this test, but 5 and 10mg/kg caused a marked decrease in the algesiometric score as compared to the control group. Temporal evolu- tion showed that morphine (10 mg/kg) produced a highly significant and continuous pain relief during the whole 30 min observation period (Fig. 1).

Biceps femoris nociceptive reflex

The amplitude of the R-Ill bioelectric EMG signal monitored 150-450 msec after the nociceptive stimuli, decreased when a central analgesic drug such as morphine was administered to the rat (Fig. 2) and could be expressed as a percentage mean of the control amplitude measured in a fixed time period (see Table 2). This is also seen in the histogram where the temporal course of the analgesia is recorded (Fig. 3). Morphine in a single dose of 10mg/kg decreased the signal amplitude, which remained diminished for more than 1 hr. This was accompanied by an evident decrease in the amplitude and frequency of respiratory movements. Naloxone (lmg/kg) reversed both effects, increasing the reflex electro- myographic signal and restoring respiratory activity of the rat.

KT did not show a dose-related decrease in the amplitude of the reflex in doses of 20, 40, 80 and

Table 2. Percentage amplitude in the biceps femoris R-Il l nociceptive reflex response in control rats and those subjected to differents

analgesic treatment*

Control Dose response amplitude

Drug (mg/kg) (%) + Mepacrine 20

Control

Morphine

Ketorolac

I00

1 44.5 _+ 4.1 (4) 5 34.6 + 8.6 (3)

I0 27.1 q- 4.5 (8)

20 84.7 _ 5.4 (3) 89.8 _ 3.2 (5) ns 40 87.2_+9.1(5) 79.6 + 3.6 (5) ns 80 82.1 _+6.6(6) 70.3 + 6.3 (5) ns

120 71.2 _+ 4.3 (6) 72.1 _+ 5.9 (5) ns

*All values are significantly different from control (P < 0.05) using Student's t-test.

ns: non significant vs ketorolac without previous mepacrine.

120 mg/kg i.v., and only produced a slight decrease in the control amplitude. The percentage inhibition in the signal amplitude was not modified by the previous i.v. administration of mepacrine (20 mg/kg, Table 2) and also remained unchanged after the administration of naloxone (1 mg/kg i.v.).

DISCUSSION

Our results demonstrate that KT, in both exper- imental tests, had only a moderate analgesic effect. However, morphine and other NSAIDs such as ASA and paracetamol (Hunskaar and Hole, 1987), lysine clonixinate (Bustamante et al., 1989) tested in the formalin test, or ASA and ketoprofene tested in the biceps femoris nociceptive reflex test (Wilier et al., 1977, 1987), show an analgesic efficacy significantly higher than KT at clinical equianalgesic doses.

The formalin test applied up to 30 min, is useful for testing the central antinociceptive activity which is evidenced by an early decrease in the algesiometric score at the beginning of the observation period (0-5 rain). By contrast, drugs that only exhibit anti- inflammatory properties are active only at the second half of the test period, when inflammatory processes take place. This is evident if we analyze the temporal course of analgesia of pure anti-inflammatory drugs, like dexamethasone or indomethacin (Hunskaar and Hole, 1987). Drugs such as ASA and other salicylates that possess a central component in their action (Dubas and Parker, 1971; Guilbaud et al., 1982), exhibit an analgesic activity in both early and late phases of the formalin test (Hunskaar et al., 1986). KT shows the most intense analgesic effect at the middle of the observation period (12-18 rain), indicat- ing a centrally originated analgesic effect in addition to its anti-inflammatory activity.

On the other hand, the biceps femoris nociceptive reflex is a technique that measures the activity of central analgesic acting drugs, since the stimuli are adjusted to produce only a direct depolarization of the sural nerve nociceptive fibers, without mediation of an inflammatory process. In this test KT showed a slight but significant analgesia, which was not modified by opiate antagonism nor prostaglandin synthesis inhibitors, indicating that opioid receptors or centrally araquidonic acid derivatives are not involved in this effect.

Very noticeable was the observation that KT did not show dose--effect relationships in either test, in the same way as reported by Domer (1990) in the writhing test in mice, which suggest that the doses used in this study (40-120 mg/kg) would be higher than the minimal dose producing 100% of the effect (ED~00). Thus, it would be evident that KT has a poor

Ketorolac experimental analgesia 697

analgesic effect that is limited by the ceiling effect of NSAIDs.

In our experimental models K T did not show the same analgesic potency of morphine and other opioids, as reported in some clinical trials (Brown et al., 1988; Yee et al., 1984, 1986; Fricke and Angelocci, 1987); on the contrary, our results are in agreement with recently reported clinical studies which demonstrate that K T is less effective than opioids in post-surgical pain management (Power et al., 1990; Powell et al.,

1990). With respect to the mechanism of action, our

results confirm that K T is devoid of opioid activity, since naloxone was unable to prevent or reverse the analgesia evoked by KT. This is in agreement with previous studies showing that K T does not bind to opioid receptors (Lopez et al., 1987), but is in dis- agreement with the results of Domer (1990), who postulated that K T might be acting through endo- genous opioid peptides. The role of prostaglandins must be elucidated by other techniques, since pharmacological associations of KT with mepacrine or indomethacin, administered 1 or 24 hr before, suggest that prostaglandins would not be involved in the central action of KT.

In conclusion, KT has only a poor analgesic effect in rats, which is not mediated by opioid receptors, and the central action of K T that we propose seems not to be related to centrally acting prostaglandins. Since other mechanisms have been suggested for centrally acting NSAIDs, such as the increased mem- brane potential and conductance due to increasing K ÷ permeability and decreasing chloride permeability in invertebrate neurons seen with ASA and derivatives (Levitan and Barker, 1972); and the calcium channel blocker activity of lysine clonixinate (Bustamante et al. 1989; Morales et al., 1992), experiments are in progress for testing whether K T modifies ionic fluxes in some isolated preparations using intracellular recordings.

Acknowledgements--This research was supported by Grant B-2897 from DTI, University of Chile. The authors are grateful to Dr Gianni Pinardi for his criticism during the manuscript redaction.

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