Baclofen as an analgesic

Brctin Re.wurh

Bullerin.

Vol. 5, Suppl.2, pp. 553-579. Printed in the U.S.A.

Baclofen as an Analgesic’

SAELENS, J. K., P. S. BERNARD AND D. E. WILSON. Buc/&r~ (IS un crnul~e~ic. BRAIN RES. BULL. 5: Suppl. 2, 553-557, 1980.-An antinociceptive effect of systemically administered baclofen (BF) was demonstrated in two rodent models, the phenylquinone-induced writhing and tail-flick procedures, at doses which also cause neurological impairment. These results suggests an antinociceptive effect of BF coinciding with a spinal muscle relaxant effect. The neuronal substrates acted upon by BF appear to be different from the opiates in that BF’s effects were unaltered by naloxone whereas those of morphine were markedly attenuated. Also, rats trained to discriminate morphine from saline failed to generalize BF to morphine in disc~minative stimulus studies. However, interactions between BF and opiates were observed in other experiments. Subthreshold doses of BF increased the antinociceptive potency of acutely administered morphine but failed to alter the development of tolerance to repeatedly administered morphine in mice. Further, BF markedly reduced naloxone precipitated jumping in morphine-dependent mice. Baclofen Morphine Drug discrimination

Antinociception Mouse jumping

Tail-flick

THE antispastic agent baclofen @-(4-chlorophenyl)-yaminobuty~c acid) has been shown to cause an antinociceptive effect in a number of animal modeIs by different investigators in different species (3,.5-12, 17, 181. Here we review a number of experiments in which we have further investigated the analgesic potential of baclofen. A comparison of previously reported data with these results supports the concept that baclofen exerts its antinociceptive activity by a mechanism separate from that of the endogenous opiate system and that baclofen’s antinociceptive activity can be demonstrated in at least one test system where spinal motor reflexes are not involved. METHOD

Unless otherwise indicated, male CF, (Carworth Farms) mice, 14-25 g; and male Wistar (Charles River) rats (350-500 g) were used. Animals were allowed food and water ad lib until the time of testing. nrl#s The following drugs were used: baclofen (base), morphine sulfate, naloxone hydrochloride, diazepam base, methadone hydro~hlo~de~ All doses refer to the salt or base form used. Soluble drugs were dissolved in distilled water. Insoluble drugs were suspended in 3% (w/v) colloidal cornstarch containing 5% (w/v) PEG-400 and 0.34% (w/v) Tween 80 and administered in a volume of 10 ml/kg. For intraventricular injections, baclofen and morphine were dissolved in sterile saline (adjusted to pH>5.0). The microinjection needle (26 pa), which protruded into the lateral ventricle when fully inserted, was connected to a Hamilton microliter syringe.

Phenylquinone

writhing

Pinch response

The volume of microinjection as a single bolus.

was 10 ~1, delivered by hand,

A modification of the test as described by Tabor [ 151was used with the phenylquinone prepared as in Blumberg c’t al. [2]. At various intervals after oral (PO) baclofen administration, mice received 2.5 mg/kg of a 5% aqueous ethanol solution of phenylquinone via the intraperitioneal (IP) route. Five minutes later the animals were placed in observation cages and the number of mice which did not perform a characteristic writhe during the next 10 min was recorded. Ten to twenty mice were used for each of 3 to 6 log~ithmically spaced doses at each time.

The method has been previously described [4]. A 10 set cut-off time was used. Baclofen was administered orally 30 and 60 min, and morphine was administered subcutaneously (SC) 30 min, before testing. Naloxone was administered intraperitoneally, simulatneously with either baclofen or morphine. Ten to twenty mice were used for each dose of each drug.

Mice were prescreened on a rod rotating at 16 rpm (UGO Basiie-Treadmill for mice). Only those mice which could remain on the bar for two min within three trials were used. The animals were placed on the rotating bar at intervals varying from 15 to 240 min after oral administration of baclofen. Any mouse which could not maintain itself on the rotating bar for the required two min within three trials was consid-

‘Part of these results were presented at the FASEB Meetings in Chicago. IL 1977.

Copyright

0 1980 ANKHO

International

Inc.-0361-9230/80/080553-05$01.00/O

554

SAELENS,

TABLE ABILITY

OF BACLOFEN

BERNARD

AND WILSON

1

TO PREVENT PHENYLQUINONE-INDUCED ROTOROD PERFORMANCE IN MICE

WRITHING

Time min

Phenylquinone ED,,, mg/kg PO (95%, confidence limits)

Rotorod ED,,, mgikg PO (95% confidence limits)

15 30 45 60 90 120 240

73.5 (24.3-222.6) 14.5 (12.g 16.5) 9.0 (5.4 14.9) 8.7 (6.3- 11.9) 8.5 (6.0- 12.1) 9.7 (6.7- 13.7) 31.5 (18.8- 54.5)

32.2 (25.3- 41.0) II.7 (IO/l- 13.2) 11.2 (lO.O- 12.5) 10.9 (9.7- 12.3) 12.0 (IO&- 13.1) 18.2 (15.9- 20.9) 140.6 (48.0-412.0)

AND DISRUPT

ED,,, rotorodl ED,,, phenylquinone 0.44 0.81 1.24 1.25 1.41 1.88 4.46

ED,,,‘s and 95% confidence limits determined by logit method 111.

ered to have a rotorod performance deficit. Ten mice were used for each of 4 logarithmically spaced doses at each time. Drug Discrimination

Studies

Rats weighing 215-250 g at the beginning of training were used as subjects. In order to maintain a state of food deprivation, food was restricted to 15 g each day given immediately after the session. Water was available ad lib in home cages. The behavioral apparatus consisted of a conventional operant chamber, which was housed in a sound-attenuated and fan-ventilated box. Each chamber contained two levers, one on either side and equidistant from a pellet dispensing trough. The rats were first magazine-trained and shaped to lever-press for food reinforcement. They were then trained to press the left lever when injected with morphine (10 mg/kg) and the right lever when injected with saline. Every tenth press (FR 10) on the appropriate lever resulted in the delivery of a 45 mg Noyes food pellet. Responses on the incorrect lever were recorded but did not result in the delivery of food. Training trials were carried out five days a week, according to an irregular alternating sequence of injection treatments. For each trial, responses emitted on each lever prior to the first reinforcement were recorded, as were the total responses emitted on each lever during the 15 min session. Training on this schedule continued until the rats made no more than four responses on the incorrect lever prior to the first 10 responses on the correct lever of each of 8 consecutive sessions. At this time generalization testing was begun. These tests (administration of novel agents) consisted of trial sessions separated by at least four practice sessions in which were correctly discriminated. and saline morphine Whenever a novel agent was administered, both levers could deliver reinforcement until 10 responses were completed on one of the levers and the first food pellet delivered. After the delivery of the reinforcer, pressing the other lever no longer delivered reinforcement but responses continued to be recorded. The lever which had been selected by the rat continued to deliver reinforcement on an FR 10 schedule. Prior to tests with baclofen, rats were required to generalize a muscle relaxant dose of diazepam (6 n&g) to saline, and to generalize an antinociceptive dose of methadone (10 mg/kg) to morphine indicating that they had acquired a narcotic cue which controlled their responding. Rats which SUC-

cessfully performed both generalizations were then tested with baclofen. Baclofen and diazepam were administered 45 min; and methadone and saline, 30 min prior to testing. All drugs were administered intraperitoneally to 4-6 rats. Jumping Test Male mice were used in a modification of the two-day system of Saelens et al. [13]. Morphine (20 mg/kg, SC) was administered on Day 1 at 9:00 a.m., 10:00 a.m., 11:OOa.m., 1:OOp.m. and 3:00 p.m.; and on Day 2 at 9:00 a.m. and 1l:OO a.m. At noon of the second day, the mice received various oral doses of baclofen or saline and at 1:00 p.m. the mice received 100 mg/kg, IP of naloxone. Each mouse was placed in a separate Plexiglas cylinder, 14 in. high and 10 in. in diameter. The number of mice which jumped (all 4 paws off the bottom surface) and the number of jumps during the IO min period following the administration of naloxone was recorded. Ten mice were used in each treatment group. RESULTS

Phenylquinone

Writhing and Rotorod Performance

Baclofen was effective in preventing phenyiquinoneinduced writhing and in disrupting rotorod performance in mice (Table 1). With the exception of the 120 min time period, the 95% confidence limits in the 2 systems overlapped. It is interesting to note the onset, time of peak effect and duration of effect of baclofen in the phenylquinone. test were not identical to that observed for disruption of rotorod performance suggesting that the neurological deficit induced by baclofen had a more rapid onset and shorter duration than the anti-writhing effect. Tail-Flick Response Parenteral administration of baclofen significantly elevated tail-flick latencies in mice (33.3 mg&g, PO) as did morphine (4 and 8 mg/kg, SC) as shown in Table 2. Unlike morphine (6 and 12 pg), administration of baclofen by the intraventricular route failed to significantly &ect the tailflick response at a maximally tolerated dose (6 pg). Drug Discrimination

Studies

After receiving baclofen (3 mg/kg, IP), the morphinetrained subjects responded on the saline lever with a fre-

BACLOFEN

555

AS AN ANALGESIC TABLE 2 EFFECTS OF INTRAVENTRICULAR AND PARENTERAL ADMINISTRATION OF BACLOFEN AND MORPHINE ON THE TAIL-FLICK RESPONSE IN MICE AND RATS

Av. ART from control (set) at: Species

Drug

Dose

Route

n*

15 min

30 mitt

Mouse

Baclofen

6.0 cLg

IVT*

20

- 1.8

-0.1

Mouse

Morphine

4.0 ~8

IVT

20

+2.2$

+3.1f

12.0 pg

IVT

20

+3.0$

+5.81:

Mouse

Baclofen

3.7 mgikg 11.1 mgikg 33.3 mgikg

PO PO PO

10 IO IO

+0.2 +o.b +2.6$

Mouse

Morphine

2.0 mgikg 4.0 mg!kg 8.0 mgikg

SC SC SC

10 IO 10

0.0 +3.8$ +4.3f

60

min

0.0 +1.2 +4.6$

*n=number of animals. iiVT=intraventricular. @,<0.05 by rank-sum method [14].

TABLE 4

TABLE 3 EFFECTS

OF BACLOFEN ON RATS TRAINED MORPHINE FROM SALINE

TO DISCRIMINATE

ABILITY OF NALOXONE TO PREVENT MORPHINE-INDUCED NOT BACLOFEN-INDUCED ELEVATIONS OF TAIL-FLICK LATENCIES IN MICE

BUT

Dose mgikg

Drug Morphine

IP IO

Total responses*

Dose mgikg

Route

Av. RT at 60 min

8.0 8.0

SC SC SC

4.65 set 9.30* set

Rat no.

Selected lever

Mean FFP”

A30 A34 B7 B31 B32

Morphine Morphine Morphine Morphine Morphine

10.0 10.2 10.2 10.5 11.5

764 543 1212 713 565

Vehicle Morphine Morphine + Naloxone

A30 A34 B7 B31 B32

Saline Saline Saline Saline Saline

10.0 10.0 10.2 10.0 10.8

1411 1025 2056 1593 1145

Vehicle Baclofen Bacfofen + Naloxone

*p
Baclofen

3.0

A34 B32 B31 B30

Saline Saline Saline Saline

12.0 10.0 17.0 11.0

1009 1152 468 1381

Baclofen

6.0

A34 B31 A30 B7

Saline Saline Saline Saline

10.0 10.0 10.0 11.0

762 1421 802 1166

Drug

4.47: set 10

SC

30 30

PO PO PO

10

SC

4.05 set 7.69* set 9.18* set

~FFP=Number of responses made to obtain the first food pellet. Values represent the mean of four trials for morphine and saline. Only one trial was performed with each dose of baclofen. tNumber of responses made during the entire 15 min session on an FR 10 schedule.

Parenteral administration of both baclofen (30 mgikg, PO) and morphine (8 mgikg, SC) significantly elevated tail-flick latencies in mice (Table 4) as previously shown for similar doses of both agents (Table 2). The narcotic antagonist, naloxone (IO mgikg, SC) completely prevented the elevation in tail-flick latency induced by morphine but had no effect on baclofen-induced elevations in tail-flick latency (Table 4).

quency similar to that of saline (Table 3). Similarly, a higher dose of baclofen (6 mg/kg, IP) induced responding on the saline lever but a frequency below that of saline. Two animals were treated with baclofen at 9 mglkg, IP but failed to emit any responses (data not shown).

Following repeated administration, morphine no longer elevated tail-flick latencies in mice (Table 5). Although an acute subthreshold dose of baclofen (10 mg/kg, PO) enhanced the antinociceptive effects of an acute dose of morphine (8 mgikg, IP), concomitant administration of the same

SAELENS,

556

BERNARD

AND WlLSON

TABLE 5

DISCUSSION

INABILlTY OF BACLOFEN TO PREVENT TOLERANCE TO MORPHINE IN THE MOUSE TAIL-FLICK RESPONSE

In these studies an antinociceptive effect of baclofen was demonstrated in two animal models. At the doses where a significant antinociceptive effect was present, a concomitant indication of neurological deficit was also present. The question arises of whether the antinociceptive measures were secondary to baclofen’s muscle relaxant effect. We would maintain that this is not a factor in the phenylquinone-writhing studies. Baclofen antagonized phenylquinone-induced writhing in mice having its peak effects 45 to 120 min following oral administration wherein the ED,,, ranged from 8.5-9.7 mgikg. The time effect relationship between the phenylquinone data (antinociceptive measure) and rotorod data (neurological deficit measure) were not the same leading to the wide range of time related therapeutic indices (0.44-4.46). These data suggest differential neuronal substrates for the antinociceptives and neurological deficit measures. Analogous results and interpretation have previously been reported for intraperitoneally administered baclofen in mice [3,5] where hot plate and rotorod performance were compared. As to whether the neurological deficit confounds the writhing antinociceptive measure, it should be noted that the endpoint observed in phenylquinone writhing is related to contractions of internal abdominal smooth muscle tissue in response to phenylquinone irritation, not spinal reflex mediated contractions of voluntary muscle. Baclofen is not known to interfere with such visceral pathways in animals. These observations indicate that baclofen’s antinociceptive effects in the phenylquinone writhing model are not merely secondary to its muscle relaxant actions.

Route

n*

Av. ifRT from control (set)

8 Acute 10 Acute 8 Acute

IP PO 1P

10 IO

+4.1+ +0.5

10

+6.7$

10 Acute

PO

Morphine

8 t.i.d. x 3 days + once on day 4

IP

10

-0.3

Baclofen

10 t.i.d. x 3 days + once on day 4

PO

10

-0.1

10

+0.9

Drug Morphine Baclofen Morphine + Baclofen

Morphine + Baclofen

Dose (mg/kg)

8 t.i.d.

x

3 days

IP

10 t.i.d. x 3 days + both once on day 4

*n=Number of animals. tp
[ 14).

TABLE 6 ABILITY OF BACLOFEN TO ATTENUATE NALOXONE PRECIPITATED JUMPING IN MORPHINE DEPENDENT MICE Dose of baclofen (mg/kg PO)

n*

Jumping rate

Vehicle: 1.25 2.5 5.0 10

60 10 10 10 30

22.3 25.1 8.5 4.3 0.9

*n=Number of animals. +Pooled control. $p
controls

Jumping incidence 60160 9110 6/10$ 4/10$ 3130$

in * by Fishers

exact

doses of baclofen with the repeated doses of morphine failed to attenuate the development of tolerance to morphine. Jumping

Test

Naloxone (100 mg/kg IP) precipitated characteristic jumping episodes in mice which had received 20 mg/kg SC of morphine seven times over a 26 hr period, as previously reported [13]. Single doses of baclofen, administered one hr before the naloxone challenge, were markedly effective in attenuating naloxone-precipitated jumping in morphine dependent mice (Table 6). It should be noted that two of the baclofen doses (2.5 and 5 mg/kg PO) which were effective in attenuating jumping were below the neurological deficitinducing dose (10.9 ml&g PO) as measured in the rotorod performance test (see above).

Parenteral administration of baclofen increased tail-flick latencies in mice to a degree similar to that obtainable with

morphine. Proudfit and Levy have made similar observations in rats [IO]. Following a series of tail-flick studies in animals with transections through various levels of the central nervous sytem, they concluded that baclofen’s antinociceptive effects were solely a result of an action in the brain stem. Of particular interest was that an antinociceptive effect of parenterally administered baclofen could be demonstrated in non-transected rats but not in rats with sections through the spinal cord (T,;-T,,,). This suggested that the elevated latencies caused by baclofen in the tail-flick response, which is a spinal reflex, cannot be explained by the general ability of baclofen to block other spinal reflexes. The authors further concluded that baclofen’s antinociceptive effects are unrelated to a spinal action of baclofen. Our results are not completely in accord with this latter conclusion. We found that parenteral administration of baclofen to mice elevated latencies in the tail-flick procedure. However, following intraventricular administration of baclofen (6 pg), latenties in the tail-flick response were unchanged from control values. Similar observations have been made in our laboratories in rats [9] where an antinociceptive effect was demonstrated for intraperitoneally but not intraventricularly administered baclofen using the tail-flick procedure. These data suggest that the effects of baclofen on the tail-flick response may be mediated by a spinal site not readily accessible from the cerebral ventricles. However, microinjections of baclofen into supraspinal sites in rats have resulted in antinociceptive activity when other antinociceptive measures were used [8,9]. Thus, the disparity of results in the tail-flick procedure are not due to species differences nor to an inherent inactivity of baclofen at supraspinal sites, and we are unable to explain these discrepancies. The suggestion of

BACLOFEN

557

AS AN ANALGESIC

Proudfit and Levy [lo] that baclofen does not have a spinal antinociceptive locus is also inconsistent with the marked antinociceptive effects following intrathecal administration of baclofen reported by Wilson and Yaksh 217,181 where baclofen was m~croinjected into the subarachnoid space surrounding the spinal cord of rats and cats. The neuronal substances affected by morphine and baclofen appear to be different. Whatever the neuronal pathways may be which mediate the tail-flick response, they were blocked by intraventricularly administered morphine but not baclofen. Further the ability of parenterally administered morphine to block the tail-flick response was prevented by coadministration of naloxone, a narcotic antagonist. The elevated tail-flick latencies induced by parenterally administered baclofen were unaffected by naloxone. In drug discrimination studies, rats which had been trained to discriminate morphine from saline with a high degree of accuracy chose the saline lever following administration of baclofen, suggesting that the internal cues elicited by morphine and baclofen are also different. Despite the apparent differences in sites of action of baclofen and morphine in the above systems, interactions between baclofen and morphine were observed. The antinociceptive effect of an acute dose of morphine was enhanced by an acute subthreshold dose of baclofen (Table 5). The interaction is not restricted to the tail-flick response as the results agree with the finding of Cutting and Jordan [3] who showed that baclofen enhanced the effects of morphine in the mouse hot-plate procedure in doses which had negligible muscle relaxant effects. Thus baclofen and morphine may exert their antinociceptive effects by separate but synergistic pathways. An additional interaction between

morphine and baclofen was noted. Doses of baclofen which were well below those causing neurological impairment markedly and significantly attenuated naloxone precipitated jumping in mo~hine-dependent mice. This phenomenon is unusually resistant to non-specific neurological impairment as shown by Robson et al. ff2] who reported that debilitating doses of haloperidol and styramate were ineffective in this model. Interestingly, these authors also reported that other GABAmimetic substances such as diazepam and muscimol were also active but that baclofen was the most selective relative to neurological impairment. In summary, baclofen has antinociceptive properties in a of animal models. number Coincidental with the antinociceptive effects, neurological impairment was identified at similar doses. Whether the antinociceptive measures were secondary to the neurological effects in some of these

models has been largely discounted by us for the phenylquinone writhing model, and by others [3,5] for the hot plate test. The animal data also suggest that the antinociceptive effects of baclofen result from an action independent of the endogenous opiate system. Yet these effects may be additive with the opiates. Clinical trial of the therapeutic potential of baclofen in human disorders with accompanying pain should tell us whether the antinociceptive properties of baclofen in animal models are reflected as analgesic properties in man.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the technical assistance of Ms. B. Barbaz and Mr. W. Autry for some of the tail-flick determinations and Mr. S. Roepke for the drug discrimination studies.

REFERENCES 1. Berkson, J. A statistically precise and relatively simple method of estimating the bioassay with quanta1 responses based on the logistical function. J. Am. Sfatisf. Ass. 48: 565-599. 19.53. 2. Biumberg, H., H. Dayton and P. Wolf. Counteraction of narcotic antagonist analgesics by the narcotic antagonist naloxone. Prot. SW. exp. Bid. Mrd. 123: 755-758. 1966. 3. Cutting, D. A. and C. C. Jordon. Alternative approaches to analgesia: Baciofen as a model compound. Br. J. Phnrmuc~. 54: 171-179, 1975. 4. Granat, F. R. and J. K. Saelens. Effect of stimulus intensity on the potency of some analgetic agents. Archs inf. Pharmc~&v~. 205: 52-60, 1973. 5. Levy, R. A. and H. K. Proudfit. Separation of analgesic and motor effects of Lioresal. Pharmudogist 18: 174, 1976. 6. Levy, R. A. and H. K. Proudfit. The analgesic action of baclofen (~-{4-chlorophenyl)-~-aminobutyric acid. f. Phrrrn-n,clc,.202: 437-445, 1977. 7. Levy, R. A. and H. K. Proudfit. Analgesia produced by mictoinjection of baclofen and morphine at brain stem sites. Err. J. Phrrrmoc. 57: 43-55, 1979. 8. Liebman, J. M. and G. Pastor. Analgesia elicited by intracerebral microinjections of baclofen and muscimol. Sot. Neltrosc?. Ahstr. 4: 461, 1978. 9. Liebman, J. M. and G. Pastor. Antinociceptive effects of baclofen and muscimol upon intraventricular ad-ministration. Submitted for publication in EUT. J. P~r~~rm~~~.

IO. Proudfit, H. K. and R. A. Levy. Delimitation of neuronal substrates necessary for the analgesic action of baclofen and morphine. EMT,1. Phcrrmcrc. 47: 159-166, 1978. II. Proudfit, H. K. and R. A. Levy. Location of neuronal substrates involved in the analgesic action of baclofen. F&r Prot,. 36: 993, 1977. 12. Robson, R. D., J. K. Saeiens and D. Wilson. Suppression of morphine withdrawal symptoms of baclofen fBF)-(Lioresal”,). Fdn Pror. 36: 1025, 1977. 13. Saelens, J. K., F. R. Granat and W. K. Sawyer. The mouse jumping test-a simple screening method to estimate the physical dependence capacity of analgesics. Ar~hs itzr. Phtrvnlcrr,o&/I. 190: 213-218, 1971. 24. Siegel, S. Nonpcrrtrmcak Sttrtisric~s. New York: McGraw-Hill, 1976. f5. Taber, R. 1.. P. D. Greenhouse and S. Irwin. Inhibition of phenylquinone-induced writhing by narcotic antagonists. Nntifr~ 204: 189-190, 1964. 16. Wilcoxon, F. and R. Wilcox. Some Rrrpid Appro.rinzcrrc Strrtisticurl Procdrtrc,.v. Pearl River, New York: Lederle Laboratories, 1964. 17. Wilson, P. R. and T. L. Yaksh. Baclofen is antinociceptive in the spinal intrathecal space of animals. Cr. J. Phtrrm~c~. 51: 323-330. 1978. IS. Wilson,‘P. R. and T. L. Yaksh. Studies on the antinociceptive effects of int~thecai baclofen in the rat and cat. Srtc.. Ncmn~ci. Abstr. 4: 436. 1978.