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Antinociceptive activity of clonidine in the mouse, rat and dog
Life Sciences, Vol. 31, pp. Printed in the U.S.A.
Pergamon Press
1123-1132
ANTINOCICEPTIVE ACTIVITY OF CLONIDINE THE MOUSE, RAT AND DOG Malcolm
Department Greenford
Skingle, Michael
of Pharmacology, Road, Greenford,
Ann G Hayes B Tyers
IN
and
Glaxo Group Research Ltd Middlesex UK UB6 OHE
(Received in final form June
30,
1982)
Summary The antinociceptive activities of clonidine have been determined against several qualitatively different noxious stimuli in the mouse, rat and dog. In these tests clonidine given subcutaneously was 6 to 7 times more potent than morphine. Both clonidine and morphine were more potent against responses to pressure and chemical nociceptive stimuli than against responses to heat induced nociception or that induced by electrical tail stimulation. However, unlike morphine the effects of clonidine in these latter tests were only seen at doses that also caused sedation and so these animals were less able to respond to the nociceptive stimuli. In contrast in pressure, chemical and tooth pulp stimulation tests clonidine produced increases in nociceptive thresholds at doses which caused no overt signs of behavioural depression. Comparisons of the relative potencies of clonidine and the less lipophilic analogue 4-hydroxyclonidine given subcutaneously and intracerebroventricularly indicate that clonidine induced antinociception is predominantly centrally mediated. However, a peripheral component may also be present in the inhibition of acetylcholine-induced abdominal constriction in the mouse. The o -adrenoceptor agonist clonidine possesses antihypertensive activity in anima 12 s (1) and man (2). More recently clonidine has been used in the treatment of migraine (3) and alleviation of the withdrawal syndrome in opiate and alcohol dependence (4, 5). In the rat and mouse, clonidine possesses potent antinociceptive activity (6, 7, 8). These latter studies differ from each other with respect to the antinociceptive potencies of clonidine. However, because different strains of mice and rats were used these studies are not strictly comparable. There is no clear evidence for an analgesic action in man. In animals, antinociceptive tests vary in their sensitivities to centrally-acting analgesic drugs. This is particularly evident for opiates (9, 10) and capsaicin (11). The present study was carried out to determine the antinociceptive activities of clonidine against qualitatively different nociceptive stim,uli (chemical, pressure, heat and electrical tooth-pulp stimulation) in the mouse, rat and dog. In these studies the same strain of mouse or rat was used to obtain comparable data in the different tests used.
0024-3205/82/111123-10$03.00/O Copyright (c) 1982 Pergamon Press Ltd.
1124
Clonidine
Antinociception
Vol.
31,
No.
11,
1982
Methods Antinociceptive
Tests
The methods used to evaluate antinociceptive activity were selected to include tests which employ different types of noxious stimuli, ie chemical, heat, pressure and electrical stimulation. Acetylcholine - induced abdominal constriction test in the mouse Tests were carried out to determine the inhibitory effects of drugs against abdominal constrictions induced by acetylcholine 3mg/kg ip. The number of abdominal constrictions occuring in the first 5 minutes thereafter was recorded. An abdominal constriction was defined as a contraction of the abdominal muscles accompanied by an extension of the hind limbs. The ED50 value was taken as the dose of test drug which reduced the number of abdominal constrictions to 50% of that occurring in placebo-treated mice. Hot-plate test in the mouse In the hot plate test, re+action times of mice placed on a+cop$er plate heated to a mean (- range) temperature of 55 -0.2 C were determined. A ‘forepaw lick’ was taken as the nociceptive response at which time the animals were rapidly removed from the hot plate; 60s was taken as the maximum reaction time. The ED value was arbitrarily defined as the dose of test drug capable of e5pevating the reaction time to twice that determined for placebo-treated animals. Tail-immersion test in the mouse and rat The tail-immersion test’was essentially the same for both mouse and rat tests. Each animal was held gently but firmly and its tail was immersed in a hot water bath maintained at 502 0.2’C. The nociceptive reaction latency &as recorded when the rodent ‘flicked’ or withdrew its tail from the water bath. The ED5o value was defined as for the hot-plate test. Paw pressure test in the rat The effects of test drugs against a noxious pressure stimulus applied to the hindpaws of the rat were determined using an ‘Analgesymeter’ (Ugo Basile, Milan). The nociceptive response wes usually a shrill vocalisation, which was clearly distinguishable from that which often occurred with normal handling, or a strong attempt to withdraw the value was arbitrarily defined as the dose of test drug Ftzt. z&Y# e nociceptive threshold by 1OOg above the mean threshold value for placebo-treated rats. The formalin test in the rat The method for the ‘formalin test’ has oreviouslv been described bv Dubuisson and Dennis (12). An injection of 0.05m1 5% formalin was injected subcutaneously into a forepaw of the rat 15 minutes after the rat had received either test drug or placebo. The response of the animal was rated according to objective behavioural criteria, which were constantly monitored for up to 1 hour after formalin administration. Nociceptive rating curves were plotted against time for each treatment group, with each point representing the mean nociceptive rating for each 3 min block. The ED5o value was defined as the dose of test drug capable of reducing the nociceptive rating score to 50% of the mean nociceptive rating occurring in placebotreated rats.
vol.
31,
No.
11,
Clonidine
1982
1125
Antinociception
Electrical tail stimulation in the rat The electrical tail stimulation method was similar to that described by Paalzow (13), except that the tail was stimulated through two external ring electrodes 15mm apart instead of using intracutaneous Electrode jelly was applied to the two electrodes to electrodes. ensure good electrical contact between electrode and tail. By increasing the voltage in steps of O.lV it was possible to determine 3 types of response: i) The motor response, when the tail flicked on stimulation, ii) The vocalisation response, when the rat vocalised for the duration of the stimulus, and iii) the vocalisation after discharge, when vocalisation continued after withdrawal of the stimulus. The voltages eliciting these 3 responses were determined in naive and drug-treated rats. Dental pulp stimulation in the conscious doq Dental pulp stimulation in the conscious Beagle dog has previously been described by Skingle and Tyers (14). In brief, two electrodes were implanted under general anaesthesia into the dentine of one of the upper canine teeth. The dogs were ready for testing 7-10 days post-operatively. The nociceptive threshold was taken as the stimulation voltage eliciting a characteristic response in each dog. Typical responses were licking, chewing or a slight head movement. The antinociceptive activity was expressed as the percentage increase in stimulus voltage required to restore the response compared with the predrug control threshold. Tests were carried out twice a week using a cross-over design. A colony of seven dogs was used. Measurement
of sedation
Clonidine-induced sedation in the rat was determined using an accelerating rotarod similar to that described by Jones and Roberts (23). The rotarod comprised 6 sections and accelerated linearly from O-50 rev/min over a 5 min period. The latencies (s) for each of the rats to fall from the rod was Rats were dosed subcutaneously with clonidine, recorded automatically. 0.03-3.0 mg/kg SC, or 0.9% w/v NaCl, 30 min prior to testing. The sedative activity (EDGE value) was defined as the dose of clonidine which reduced the reaction latency to 50% of the mean latency achieved by control rats. Nociceptive pressure thresholds were also determined in those rats prior to testing on the rotarod. The order of testing was always the same. Animals
and Experimental
Desiqn
Tests in the mouse (male, CRH, 18-22g) and rat (male, AH random bred Hooded, 35-709) were all carried out under essentially the same experimental conditions. Individual tests were carried out using dose-groups of 6 animals. Data for the calculation of ED5o values were accumulated from 2 or 3 individual tests carried out on different days (c7 days apart), so that the final dose-groups comprised 12 or 18 animals respectively. To eliminate cage interactions the animals were randomized into different cages so that each cage contained animals receiving different treatments. Because tests normally lasted a full working day this latter procedure also randomized treatments with respect to time of day and thus eliminated any bias associated with temporal cycles of pain sensitivity. Animals and drug solutions were colour coded such that the operators were unaware of the treatments the animals were receiving. In tooth-pulp stimulation experiments, Operators were unaware of the treatments
Beagle dogs (9-15kg) each dog received.
were
used.
1126
Clonidlne Antinoclception
Vol. 31, No. Ii, 1982
Drug Administration In each test, clonidine was compared with morphine. All tests included a placebo control in which 0.9% w/v NaCI was given in the same dose volume as the test drugs. Drugs were dissolved in saline and injected either subcutaneously (sc), in dose volumes of 0.2ml/20g and 0.4ml/100g in the mouse and rat, respectively, or intracerebroventricularly (icv), in dose volumes of 5ul/mouse and 10ul/rat. ICV injections were delivered into the lateral ventricle in the conscious animal according to the methods of Brittain and Handley (15) in the mouse, and Popick (16) in the rat. Subcutaneous injections in the dog were given in a dose volume of 0.1ml/kg. Antinociceptive activities (EOc,n) were determined at 15 and 30 rain after icy and sc injection, respectiv~Ty, as prior experiments had shown these to be the times of peak effect. Druqs The following drugs were used: morphine hydrochloride (Sterling Winthrop): clonidine hydrochloride and 4-hydroxyclonidine hydrobromide (BoehringerIngelheim); acetylcholine chloride (Sigma). Doses refer to the free bases of drugs used. RESULTS Acetylcholine - induced abdominal constriction test in the mouse In the conscious mouse clonidine, 0.003-0.1mglkg sc and 0.15-3.0ug/mouse icv and morphine, 0.1-2.0mg/kg sc and 0.001-0.03ug/mouse, caused significant and dose-dependant reductions in the number of acetylcholineinduced abdominal constrictions. Similarly, 4-hydroxyclonidine, the major metabolite of clonidine, in the dose-ranges 0.9-8.1mg/kg so, 1-27ug/mouse icv also reduced the number of acetylcholine-induced abdominal constrictions. Dose-response lines were linear and parallel.
In these tests, when given subcutaneously, clonidine was 28 times more potent than morphine and 200 times more potent that 4-hydroxyclonidine (Table 1). In contrast, when given icv, clonidine was 4 times less potent that morphine and only 6.5 times more potent that 4-hydroxyclonidine. It is also interesting to note that when the iev data are expressed in terms of body weight, clonidine was about equipotent with sc dosing while 4hydroxyclonidine and morphine were markedly less potent by the subcutaneous route. TABLE 1 Antinociceptive potencies of clonidine~ 4-hydroxyclonidine and morphine in inhibiting acetylcholine-induced abdominal constrictions in the mouse Antinociceptive limits) mg/Kg Drug
activity
ED50
(95%
confidence
Subcutaneous
Intracerebroventricular
Clonidine
0.016 (0.003-0.11)
0.015 (0.002-0.084)
4-hydroxyclonidine
3.29 (0.62-30.0)
0.i0 (0.016-0.56)
Morphine
0.45 (0.22-0.85)
0.0035 (0.0008-0.011)
Vol. 31, No. II, 1982
Clonidine Antinociception
1127
Hot-plate and tail-immersion tests in the mouse and rat In the hot-plate and tail-immersion tests in the mouse and rat both clonidine and morphine given sc caused dose-dependant increases in nociceptive reaction latencies. Dose-response lines were linear and parallel. In the mouse clonidine was 6-7 times more potent than morphine (Table 2) but in the rat these drugs were approximately equipotent. It is also interesting that while in the mouse both drugs were more potent in the tail-immersion test than in the hot-plate test, in the rat the converse was found, especially for clonidine. TABLE 2 Antinociceptive potencies of clonidine and morphine in the hot-plate and tail immersion tests in the mouse and rat Antinociceptive a c t i v i t y ED50 (95% confidence limits) mg/Kg S.C. MOUSE
DRUG
Hot-plate test
Clonidine Morphine
RAT
Tail-immersion test
0.29
0.077
Hot-plate test 0.79
Tail-immersion test 2.69
(0.03-2.6)
(0.004-1.35)
(0.13-4.02)
(0.64-11.66)
1.6 (0.5-5.1)
0.53 (0.15-1.87)
0.84 (0.49-1.41)
1.22 (0.29-5.41)
Paw pressure test in the weanling rat In the paw pressure test in the weanling rat clonidine, 0.0]-O.Smg/kg sc and 0.025-O.2mg/kg icv, both increased nociceptive pressure thresholds in a dose-dependant manner (Fig 1). Morphine, 0.]-3.0mg/kg sc also caused dose-dependant increases in nociceptive thresholds. Following subcutaneous administration of clonidine the peak antinociceptive effect occured 30 minutes after drug administration and significant doserelated effects were still apparent after 90 minutes. The antinociceptive activities (EDen) of subcutaneously administered clonidine and morphine against pressu~r~-induced nociception were 0.08 (0.05-0.13) and 0.4 (0.20.7)mg/kg sc respectively. Thus, clonidine was 5 times more potent than morphine in this model. The antinociceptive potency of clonidine (EDsn = 0.047 (0.017-0.12)mg/kg icv) was only slightly greater when delivered i 5 t o the lateral ventricle compared to the subcutaneous route. Following icv dosing there was a very rapid (2 minute) onset of antinociceptive effect which reached maximum 10-15 minutes after dosing. Formalin t e s t in the rat In the rat clonidine, 0.05-0.2mg/kg sc (Fig 2) and morphine, 0.3-3.0mg/kg sc, reduced the nociceptive response to a subplantar injection of formalin in a dose-dependant manner. Peak antinociceptive effects for c]onidine were reached at about 40 rain after dosing and the effect of the highest dose was still maximal 90 min after dosing. The antinociceptive activities (ED~,n) for clonidine and morphine in the formalin test were 0.14 (0.04-0.2~Y and 0.8(0.23-1.36)mg/kg sc respectively.
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Clonidine
Antinociception
Vol.
31, No. 11, 1982
IMRACERESROVENTRICUUR
Fig 1 Effects of clonidine nociceptive pressure
(II-12)
qiven subcutaneously and intracerebroventricularly thresholds in the weanlinq rat
on
3.0
0
Placsbo
0.8 mg/Kg s;,c. l 0.1 " . 0.2 " "
2.5
l
h4
d 2.0 .-P z! c 1.5 .g Q 2 1.0
0.5.
I Fornhl injection
I
15
I
30
1
4
45
60
Time (mind
Fig 2 The antinociceptive effects of clonidine nociceptive response to a subplantar injection
qiven subcutaneously of formalin
on
the
Vol. 31, No. ii, 1982
Clonldine Antinociceptlon
1129
Electrical stimulation of the tail in the rat In the tail electrical stimulation test in the rat the voltages eliciting each of the nociceptive responses were increased by clonidine, O.:)125-1.25mg/kg se, and morphine, 1-9mg/kg sc (Figure 3). In this test, clonidine was approximately three times more potent than morphine.
• V0calisation after discharge (VAD); • Vocalisation response (VR); • Motor response MR) (n=12)
m~m
/
~-m
Control VAi) threshold
. . . .
~3
z
C0n;v . t.resht= e
~ _ _ _c_o_n.-~
+ 0.3125
.
.
.
.
.
_!_h_r_ts~ - . . . . . . . . . .
~ 0.625 CLONIDINE
+ 1.25
+ 1 Dose mg/Kg s.c.
+
+
3 MORPHINE
9
Fig 3 The antinociceptive effects of c[onidine and morphine in the electrical tail stimulation test in the rat
Rotarod test in the rat
In the rotarod test in the rat clonidine, O.1-3.0mg/kg s.c and morphine 4.01G.0mg/kg so, caused dose-related decreases in reaction latencies. The EDen values for clonidine and morphine were 0.37 (0.18-0.82) mg/kg sc and 7.5"~'4.2-16.5) mg/kg sc respectively. These values were 5 and 19 times higher than the corresponding EDsFI values obtained for these drugs in paw pressure tests in the rat. Howeve~doses of elonidine higher than the EDs0 dose in the rotarod test were necessary to increase reaction latencies in the hot-plate and tail-immersion tests in the rat. Tooth-pulp stimulation in the doq in the conscious dog clonidine, 6.25-50ug/kg sc, caused significant (P 20.05) dose-dependant increases in nociceptive thresholds to electrical stimulation of the tooth pulp (Figure 4). A t the highest dose of clonidine, 50 ug/kg sc, noeiceptive thresholds were almost 30% above pre-injection values. This dose produced significant increases in nociceptive thresholds within 15 minutes. Peak antinociception occurred at 30 minutes and the duration of action was approximately 2
1130
Clonidine Antinociception
Vol. 31, No. ii, 1982
A t the highest dose marked sedation was observed in two of the clonidine-dosed dogs. Clonidine was 4-5 times more potent t h a n morphine (10) in the tooth-pulp stimulation test when administered subcutaneously,
hours. seven
30-
...
• 50 pg/kg
i
:
s.c.
:: ::
c
|
iso Time after dosing (rains) Significantly different from vehicle alone - ° p < 0 . 0 : "° p
Fig 4 Effects of cJonidine qiven subcutaneously on nociceptive thresholds to tooth-pulp stimulation in the conscious doq
DISCUSSION The results presented here show that clonidine has potent antinociceptive properties against several types of noxious stimuli. The antinociceptive models using pressure (rat paw pressure test) and chemical (formalin and abdominal constriction test) stimuli were most sensitive to the effects of clonidine and yielded the most reproducible results. Tests in which heal was the noxious stimulus were less sensitive to the effects of both clonidine and morphine. Clonidine, unlike morphine, is sedative at the same dose levels and so a c t i v i t y in these latter tests may reflect the sedative properties of the drug rather than a specific antinociceptive effect. The rat tail electrical stimulation model was also less sensitive to the effects of c]onidine than tests employing pressure or chemical nociceptive stimuli. In contrast, low doses of clonidine reduced the response to tooth-pulp stimulation in the dog at doses which had no overt effects on behaviour. The site of action of clonidine-induced antinociception is unknown, although the results presented here suggest that the effect is predominantly of central origin. This is demonstrated by comparing the relative potencies of c]onidine and the Jess lipophilic analogue 4-hydroxyclonidine when given sc and icv. Following icv dosing in the mouse clonidine was 6 to 7 times more potent than 4-hydroxyclonidine at inhibiting acetylcholine-induced abdominal constrictions. This agrees well with the
Voi. 31, No. 11, 1982
Clonidine Antinociception
1131
relative potency for these imidazolines at presynaptie e2-adrenoceptors in the pithed rat preparation (22). However, while clonidine was as potent subcutaneously as it was icv, reconfirming that it has little difficulty in passing the blood brain barrier, 4-hydroxyclonidine when given subcutaneously was 30 times less potent than when it was given icv. These results suggest that the antinociceptive effects of these drugs in the acetylcholine-induced abdominal constriction test is predominantly via a central mechanism. However, a peripheral component to the antinociceptive effect of clonidine in this test is likely as suggested by Bentley, Copeland and Starr (20,21), since clonidine was relatively much more potent than morphine in this test than in any of the other tests. It has been suggested that the antinociceptive action of clonidine is mediated entirely at spinal level (18). However, much higher intrathecal doses of clonidine are necessary to produce an antinociceptive effect (27) compared with the small doses required for icy administration. Thus the effect may be mediated, at least in part, via a spinal mechanism but the predominant site appears to be supraspinal. Possibly, clonidine-induced antiniciception is analogous to its well documented anti-hypertensive effects, in that low doses exert their effect supraspinally, and higher doses are required to exert an action at spinal sites (19). The powerful that it may evidence as properties of
antinociceptive effect of clonidine in several animal species suggests be of clinical use as a non-narcotic analgesic although there is no to its efficacy in man. However, the sedative and hypotensive the drug may limit its therapeutic usefulness as an analgesic. Acknowledqements
We would like to thank Miss Susan Purdy, Miss Julia Wilson and Mr Vrajesh Patel for their excellent technical assistance. We are also indebted to BoehringerIngelheim for their generous gifts of clonidine HCI and 4-hydroxyclonidine HBr. References
i) z) 3) 4)
5) 6) 7) 8) 9) io) 11)
12) 13) 14)
15) 16)
HOEFKE, W. and KOBINGER, W., Arzneimittel-Forsch. 16. 1038-1056 (1966) DOLLERY, C.T., DAVIES, D.S., DRAFFAN, G.H., DARGIE, H.3., DEAN, C.R., REID, 3.L., CLARE, R.A. and MURRAY, S., Clin Pharmacol Ther., 1__9,11-17 (1976) DAS, S.M., AHUJA, G.N. and NAIRAINASWAMY, A.S., Acts Neurol. Stand., 60 (/4), 214-217 (1979) GOLD, M.S., POTTASH, A.C. and KLEBER, H.D., Lancet, 1 (8220), 621 (i98i) BJORKQVIST, S.E., Aeta Psychiat. Scand (Denmark), 52 (4), 256-263 (1975) PAALZOW, G and PAALZOW, L., Naunyn-Schmiedeb. Arch Pharmacol. 2_92, i19-126 (1976) FIELDING, S., WIKLER, J., HYNES, M., SZEWCZAK, M., NOVICK, W.J. and LAL, H., J. Pharm. Exp. Ther., 207 (3), 899-905 (1978) STOCKHAUS, K. From "Problems of Drug Dependence", Nat. Acad. Sol. 355-366, (1977) TYERS, M.B., Br. 3. Pharmac., 6_99,503-512 (1980) SKINGLE, M. and TYERS, M.B., Br. 3. Pharmacol, 713, 323-327 (1980) HAYES, A.G. and TYERS, M.B., Brain Res., 189, 561-564 (1980) DUBUISSON, D. and DENNIS, S.G., Pain, 4, 161-174, (1977) PAALZOW, L., 3. Phar, Pharmac, 2._66,361-363 (1974) SKINGLE, M., and TYERS, M.B. 3. PharmacoI Methods, _2, 71-80 (1979) BIRITTAIN, R.T., and HANDLEY, S.L. 3. Physiol., Lond., 392, 805-813 (i967) POPICK, F.R., Life Sci, 18, 197-204 (1976)
1132
17) 18) 19)
Clonfdlne Antfnociceptlon
Vol. 31, No. 11, 1982
CAHUSAC, P., HAYES9 A.G., SKINGLE, M. and TYERS, M.B. Brit. 3. Pharmacol, 7_44,96)P (1981) SPAULDING, T.C., VENAFRO, 3.3., Ma., M.G. and FIELDING, S. Neuropharm, ~ 103-105 (1979) SINHA, 3.N., ATKINSON, 3.M. and SCHMITT, H. Eur. 3. Pharmacol, 24, 11)-119 (197))
20) 21) 22) 2))
BENTLEY, G.A., COPELAND, I.W., STARR~ 3. Olin. Exp. Pharmac. Physiol. _4, 405-419 (1977) BENTLEY, (3.A. and STARR, 3. Clin. ExP. Pharmacol. Physiol, 6, 702 (1979) DREW, G.M. (Unpublished) 3ONES, B.3. and ROBERTS, D.3. 3. Pharm. Pharmac., 20, 302-304 (1968)
Pergamon Press
1123-1132
ANTINOCICEPTIVE ACTIVITY OF CLONIDINE THE MOUSE, RAT AND DOG Malcolm
Department Greenford
Skingle, Michael
of Pharmacology, Road, Greenford,
Ann G Hayes B Tyers
IN
and
Glaxo Group Research Ltd Middlesex UK UB6 OHE
(Received in final form June
30,
1982)
Summary The antinociceptive activities of clonidine have been determined against several qualitatively different noxious stimuli in the mouse, rat and dog. In these tests clonidine given subcutaneously was 6 to 7 times more potent than morphine. Both clonidine and morphine were more potent against responses to pressure and chemical nociceptive stimuli than against responses to heat induced nociception or that induced by electrical tail stimulation. However, unlike morphine the effects of clonidine in these latter tests were only seen at doses that also caused sedation and so these animals were less able to respond to the nociceptive stimuli. In contrast in pressure, chemical and tooth pulp stimulation tests clonidine produced increases in nociceptive thresholds at doses which caused no overt signs of behavioural depression. Comparisons of the relative potencies of clonidine and the less lipophilic analogue 4-hydroxyclonidine given subcutaneously and intracerebroventricularly indicate that clonidine induced antinociception is predominantly centrally mediated. However, a peripheral component may also be present in the inhibition of acetylcholine-induced abdominal constriction in the mouse. The o -adrenoceptor agonist clonidine possesses antihypertensive activity in anima 12 s (1) and man (2). More recently clonidine has been used in the treatment of migraine (3) and alleviation of the withdrawal syndrome in opiate and alcohol dependence (4, 5). In the rat and mouse, clonidine possesses potent antinociceptive activity (6, 7, 8). These latter studies differ from each other with respect to the antinociceptive potencies of clonidine. However, because different strains of mice and rats were used these studies are not strictly comparable. There is no clear evidence for an analgesic action in man. In animals, antinociceptive tests vary in their sensitivities to centrally-acting analgesic drugs. This is particularly evident for opiates (9, 10) and capsaicin (11). The present study was carried out to determine the antinociceptive activities of clonidine against qualitatively different nociceptive stim,uli (chemical, pressure, heat and electrical tooth-pulp stimulation) in the mouse, rat and dog. In these studies the same strain of mouse or rat was used to obtain comparable data in the different tests used.
0024-3205/82/111123-10$03.00/O Copyright (c) 1982 Pergamon Press Ltd.
1124
Clonidine
Antinociception
Vol.
31,
No.
11,
1982
Methods Antinociceptive
Tests
The methods used to evaluate antinociceptive activity were selected to include tests which employ different types of noxious stimuli, ie chemical, heat, pressure and electrical stimulation. Acetylcholine - induced abdominal constriction test in the mouse Tests were carried out to determine the inhibitory effects of drugs against abdominal constrictions induced by acetylcholine 3mg/kg ip. The number of abdominal constrictions occuring in the first 5 minutes thereafter was recorded. An abdominal constriction was defined as a contraction of the abdominal muscles accompanied by an extension of the hind limbs. The ED50 value was taken as the dose of test drug which reduced the number of abdominal constrictions to 50% of that occurring in placebo-treated mice. Hot-plate test in the mouse In the hot plate test, re+action times of mice placed on a+cop$er plate heated to a mean (- range) temperature of 55 -0.2 C were determined. A ‘forepaw lick’ was taken as the nociceptive response at which time the animals were rapidly removed from the hot plate; 60s was taken as the maximum reaction time. The ED value was arbitrarily defined as the dose of test drug capable of e5pevating the reaction time to twice that determined for placebo-treated animals. Tail-immersion test in the mouse and rat The tail-immersion test’was essentially the same for both mouse and rat tests. Each animal was held gently but firmly and its tail was immersed in a hot water bath maintained at 502 0.2’C. The nociceptive reaction latency &as recorded when the rodent ‘flicked’ or withdrew its tail from the water bath. The ED5o value was defined as for the hot-plate test. Paw pressure test in the rat The effects of test drugs against a noxious pressure stimulus applied to the hindpaws of the rat were determined using an ‘Analgesymeter’ (Ugo Basile, Milan). The nociceptive response wes usually a shrill vocalisation, which was clearly distinguishable from that which often occurred with normal handling, or a strong attempt to withdraw the value was arbitrarily defined as the dose of test drug Ftzt. z&Y# e nociceptive threshold by 1OOg above the mean threshold value for placebo-treated rats. The formalin test in the rat The method for the ‘formalin test’ has oreviouslv been described bv Dubuisson and Dennis (12). An injection of 0.05m1 5% formalin was injected subcutaneously into a forepaw of the rat 15 minutes after the rat had received either test drug or placebo. The response of the animal was rated according to objective behavioural criteria, which were constantly monitored for up to 1 hour after formalin administration. Nociceptive rating curves were plotted against time for each treatment group, with each point representing the mean nociceptive rating for each 3 min block. The ED5o value was defined as the dose of test drug capable of reducing the nociceptive rating score to 50% of the mean nociceptive rating occurring in placebotreated rats.
vol.
31,
No.
11,
Clonidine
1982
1125
Antinociception
Electrical tail stimulation in the rat The electrical tail stimulation method was similar to that described by Paalzow (13), except that the tail was stimulated through two external ring electrodes 15mm apart instead of using intracutaneous Electrode jelly was applied to the two electrodes to electrodes. ensure good electrical contact between electrode and tail. By increasing the voltage in steps of O.lV it was possible to determine 3 types of response: i) The motor response, when the tail flicked on stimulation, ii) The vocalisation response, when the rat vocalised for the duration of the stimulus, and iii) the vocalisation after discharge, when vocalisation continued after withdrawal of the stimulus. The voltages eliciting these 3 responses were determined in naive and drug-treated rats. Dental pulp stimulation in the conscious doq Dental pulp stimulation in the conscious Beagle dog has previously been described by Skingle and Tyers (14). In brief, two electrodes were implanted under general anaesthesia into the dentine of one of the upper canine teeth. The dogs were ready for testing 7-10 days post-operatively. The nociceptive threshold was taken as the stimulation voltage eliciting a characteristic response in each dog. Typical responses were licking, chewing or a slight head movement. The antinociceptive activity was expressed as the percentage increase in stimulus voltage required to restore the response compared with the predrug control threshold. Tests were carried out twice a week using a cross-over design. A colony of seven dogs was used. Measurement
of sedation
Clonidine-induced sedation in the rat was determined using an accelerating rotarod similar to that described by Jones and Roberts (23). The rotarod comprised 6 sections and accelerated linearly from O-50 rev/min over a 5 min period. The latencies (s) for each of the rats to fall from the rod was Rats were dosed subcutaneously with clonidine, recorded automatically. 0.03-3.0 mg/kg SC, or 0.9% w/v NaCl, 30 min prior to testing. The sedative activity (EDGE value) was defined as the dose of clonidine which reduced the reaction latency to 50% of the mean latency achieved by control rats. Nociceptive pressure thresholds were also determined in those rats prior to testing on the rotarod. The order of testing was always the same. Animals
and Experimental
Desiqn
Tests in the mouse (male, CRH, 18-22g) and rat (male, AH random bred Hooded, 35-709) were all carried out under essentially the same experimental conditions. Individual tests were carried out using dose-groups of 6 animals. Data for the calculation of ED5o values were accumulated from 2 or 3 individual tests carried out on different days (c7 days apart), so that the final dose-groups comprised 12 or 18 animals respectively. To eliminate cage interactions the animals were randomized into different cages so that each cage contained animals receiving different treatments. Because tests normally lasted a full working day this latter procedure also randomized treatments with respect to time of day and thus eliminated any bias associated with temporal cycles of pain sensitivity. Animals and drug solutions were colour coded such that the operators were unaware of the treatments the animals were receiving. In tooth-pulp stimulation experiments, Operators were unaware of the treatments
Beagle dogs (9-15kg) each dog received.
were
used.
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Clonidlne Antinoclception
Vol. 31, No. Ii, 1982
Drug Administration In each test, clonidine was compared with morphine. All tests included a placebo control in which 0.9% w/v NaCI was given in the same dose volume as the test drugs. Drugs were dissolved in saline and injected either subcutaneously (sc), in dose volumes of 0.2ml/20g and 0.4ml/100g in the mouse and rat, respectively, or intracerebroventricularly (icv), in dose volumes of 5ul/mouse and 10ul/rat. ICV injections were delivered into the lateral ventricle in the conscious animal according to the methods of Brittain and Handley (15) in the mouse, and Popick (16) in the rat. Subcutaneous injections in the dog were given in a dose volume of 0.1ml/kg. Antinociceptive activities (EOc,n) were determined at 15 and 30 rain after icy and sc injection, respectiv~Ty, as prior experiments had shown these to be the times of peak effect. Druqs The following drugs were used: morphine hydrochloride (Sterling Winthrop): clonidine hydrochloride and 4-hydroxyclonidine hydrobromide (BoehringerIngelheim); acetylcholine chloride (Sigma). Doses refer to the free bases of drugs used. RESULTS Acetylcholine - induced abdominal constriction test in the mouse In the conscious mouse clonidine, 0.003-0.1mglkg sc and 0.15-3.0ug/mouse icv and morphine, 0.1-2.0mg/kg sc and 0.001-0.03ug/mouse, caused significant and dose-dependant reductions in the number of acetylcholineinduced abdominal constrictions. Similarly, 4-hydroxyclonidine, the major metabolite of clonidine, in the dose-ranges 0.9-8.1mg/kg so, 1-27ug/mouse icv also reduced the number of acetylcholine-induced abdominal constrictions. Dose-response lines were linear and parallel.
In these tests, when given subcutaneously, clonidine was 28 times more potent than morphine and 200 times more potent that 4-hydroxyclonidine (Table 1). In contrast, when given icv, clonidine was 4 times less potent that morphine and only 6.5 times more potent that 4-hydroxyclonidine. It is also interesting to note that when the iev data are expressed in terms of body weight, clonidine was about equipotent with sc dosing while 4hydroxyclonidine and morphine were markedly less potent by the subcutaneous route. TABLE 1 Antinociceptive potencies of clonidine~ 4-hydroxyclonidine and morphine in inhibiting acetylcholine-induced abdominal constrictions in the mouse Antinociceptive limits) mg/Kg Drug
activity
ED50
(95%
confidence
Subcutaneous
Intracerebroventricular
Clonidine
0.016 (0.003-0.11)
0.015 (0.002-0.084)
4-hydroxyclonidine
3.29 (0.62-30.0)
0.i0 (0.016-0.56)
Morphine
0.45 (0.22-0.85)
0.0035 (0.0008-0.011)
Vol. 31, No. II, 1982
Clonidine Antinociception
1127
Hot-plate and tail-immersion tests in the mouse and rat In the hot-plate and tail-immersion tests in the mouse and rat both clonidine and morphine given sc caused dose-dependant increases in nociceptive reaction latencies. Dose-response lines were linear and parallel. In the mouse clonidine was 6-7 times more potent than morphine (Table 2) but in the rat these drugs were approximately equipotent. It is also interesting that while in the mouse both drugs were more potent in the tail-immersion test than in the hot-plate test, in the rat the converse was found, especially for clonidine. TABLE 2 Antinociceptive potencies of clonidine and morphine in the hot-plate and tail immersion tests in the mouse and rat Antinociceptive a c t i v i t y ED50 (95% confidence limits) mg/Kg S.C. MOUSE
DRUG
Hot-plate test
Clonidine Morphine
RAT
Tail-immersion test
0.29
0.077
Hot-plate test 0.79
Tail-immersion test 2.69
(0.03-2.6)
(0.004-1.35)
(0.13-4.02)
(0.64-11.66)
1.6 (0.5-5.1)
0.53 (0.15-1.87)
0.84 (0.49-1.41)
1.22 (0.29-5.41)
Paw pressure test in the weanling rat In the paw pressure test in the weanling rat clonidine, 0.0]-O.Smg/kg sc and 0.025-O.2mg/kg icv, both increased nociceptive pressure thresholds in a dose-dependant manner (Fig 1). Morphine, 0.]-3.0mg/kg sc also caused dose-dependant increases in nociceptive thresholds. Following subcutaneous administration of clonidine the peak antinociceptive effect occured 30 minutes after drug administration and significant doserelated effects were still apparent after 90 minutes. The antinociceptive activities (EDen) of subcutaneously administered clonidine and morphine against pressu~r~-induced nociception were 0.08 (0.05-0.13) and 0.4 (0.20.7)mg/kg sc respectively. Thus, clonidine was 5 times more potent than morphine in this model. The antinociceptive potency of clonidine (EDsn = 0.047 (0.017-0.12)mg/kg icv) was only slightly greater when delivered i 5 t o the lateral ventricle compared to the subcutaneous route. Following icv dosing there was a very rapid (2 minute) onset of antinociceptive effect which reached maximum 10-15 minutes after dosing. Formalin t e s t in the rat In the rat clonidine, 0.05-0.2mg/kg sc (Fig 2) and morphine, 0.3-3.0mg/kg sc, reduced the nociceptive response to a subplantar injection of formalin in a dose-dependant manner. Peak antinociceptive effects for c]onidine were reached at about 40 rain after dosing and the effect of the highest dose was still maximal 90 min after dosing. The antinociceptive activities (ED~,n) for clonidine and morphine in the formalin test were 0.14 (0.04-0.2~Y and 0.8(0.23-1.36)mg/kg sc respectively.
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Clonidine
Antinociception
Vol.
31, No. 11, 1982
IMRACERESROVENTRICUUR
Fig 1 Effects of clonidine nociceptive pressure
(II-12)
qiven subcutaneously and intracerebroventricularly thresholds in the weanlinq rat
on
3.0
0
Placsbo
0.8 mg/Kg s;,c. l 0.1 " . 0.2 " "
2.5
l
h4
d 2.0 .-P z! c 1.5 .g Q 2 1.0
0.5.
I Fornhl injection
I
15
I
30
1
4
45
60
Time (mind
Fig 2 The antinociceptive effects of clonidine nociceptive response to a subplantar injection
qiven subcutaneously of formalin
on
the
Vol. 31, No. ii, 1982
Clonldine Antinociceptlon
1129
Electrical stimulation of the tail in the rat In the tail electrical stimulation test in the rat the voltages eliciting each of the nociceptive responses were increased by clonidine, O.:)125-1.25mg/kg se, and morphine, 1-9mg/kg sc (Figure 3). In this test, clonidine was approximately three times more potent than morphine.
• V0calisation after discharge (VAD); • Vocalisation response (VR); • Motor response MR) (n=12)
m~m
/
~-m
Control VAi) threshold
. . . .
~3
z
C0n;v . t.resht= e
~ _ _ _c_o_n.-~
+ 0.3125
.
.
.
.
.
_!_h_r_ts~ - . . . . . . . . . .
~ 0.625 CLONIDINE
+ 1.25
+ 1 Dose mg/Kg s.c.
+
+
3 MORPHINE
9
Fig 3 The antinociceptive effects of c[onidine and morphine in the electrical tail stimulation test in the rat
Rotarod test in the rat
In the rotarod test in the rat clonidine, O.1-3.0mg/kg s.c and morphine 4.01G.0mg/kg so, caused dose-related decreases in reaction latencies. The EDen values for clonidine and morphine were 0.37 (0.18-0.82) mg/kg sc and 7.5"~'4.2-16.5) mg/kg sc respectively. These values were 5 and 19 times higher than the corresponding EDsFI values obtained for these drugs in paw pressure tests in the rat. Howeve~doses of elonidine higher than the EDs0 dose in the rotarod test were necessary to increase reaction latencies in the hot-plate and tail-immersion tests in the rat. Tooth-pulp stimulation in the doq in the conscious dog clonidine, 6.25-50ug/kg sc, caused significant (P 20.05) dose-dependant increases in nociceptive thresholds to electrical stimulation of the tooth pulp (Figure 4). A t the highest dose of clonidine, 50 ug/kg sc, noeiceptive thresholds were almost 30% above pre-injection values. This dose produced significant increases in nociceptive thresholds within 15 minutes. Peak antinociception occurred at 30 minutes and the duration of action was approximately 2
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Clonidine Antinociception
Vol. 31, No. ii, 1982
A t the highest dose marked sedation was observed in two of the clonidine-dosed dogs. Clonidine was 4-5 times more potent t h a n morphine (10) in the tooth-pulp stimulation test when administered subcutaneously,
hours. seven
30-
...
• 50 pg/kg
i
:
s.c.
:: ::
c
|
iso Time after dosing (rains) Significantly different from vehicle alone - ° p < 0 . 0 : "° p
Fig 4 Effects of cJonidine qiven subcutaneously on nociceptive thresholds to tooth-pulp stimulation in the conscious doq
DISCUSSION The results presented here show that clonidine has potent antinociceptive properties against several types of noxious stimuli. The antinociceptive models using pressure (rat paw pressure test) and chemical (formalin and abdominal constriction test) stimuli were most sensitive to the effects of clonidine and yielded the most reproducible results. Tests in which heal was the noxious stimulus were less sensitive to the effects of both clonidine and morphine. Clonidine, unlike morphine, is sedative at the same dose levels and so a c t i v i t y in these latter tests may reflect the sedative properties of the drug rather than a specific antinociceptive effect. The rat tail electrical stimulation model was also less sensitive to the effects of c]onidine than tests employing pressure or chemical nociceptive stimuli. In contrast, low doses of clonidine reduced the response to tooth-pulp stimulation in the dog at doses which had no overt effects on behaviour. The site of action of clonidine-induced antinociception is unknown, although the results presented here suggest that the effect is predominantly of central origin. This is demonstrated by comparing the relative potencies of c]onidine and the Jess lipophilic analogue 4-hydroxyclonidine when given sc and icv. Following icv dosing in the mouse clonidine was 6 to 7 times more potent than 4-hydroxyclonidine at inhibiting acetylcholine-induced abdominal constrictions. This agrees well with the
Voi. 31, No. 11, 1982
Clonidine Antinociception
1131
relative potency for these imidazolines at presynaptie e2-adrenoceptors in the pithed rat preparation (22). However, while clonidine was as potent subcutaneously as it was icv, reconfirming that it has little difficulty in passing the blood brain barrier, 4-hydroxyclonidine when given subcutaneously was 30 times less potent than when it was given icv. These results suggest that the antinociceptive effects of these drugs in the acetylcholine-induced abdominal constriction test is predominantly via a central mechanism. However, a peripheral component to the antinociceptive effect of clonidine in this test is likely as suggested by Bentley, Copeland and Starr (20,21), since clonidine was relatively much more potent than morphine in this test than in any of the other tests. It has been suggested that the antinociceptive action of clonidine is mediated entirely at spinal level (18). However, much higher intrathecal doses of clonidine are necessary to produce an antinociceptive effect (27) compared with the small doses required for icy administration. Thus the effect may be mediated, at least in part, via a spinal mechanism but the predominant site appears to be supraspinal. Possibly, clonidine-induced antiniciception is analogous to its well documented anti-hypertensive effects, in that low doses exert their effect supraspinally, and higher doses are required to exert an action at spinal sites (19). The powerful that it may evidence as properties of
antinociceptive effect of clonidine in several animal species suggests be of clinical use as a non-narcotic analgesic although there is no to its efficacy in man. However, the sedative and hypotensive the drug may limit its therapeutic usefulness as an analgesic. Acknowledqements
We would like to thank Miss Susan Purdy, Miss Julia Wilson and Mr Vrajesh Patel for their excellent technical assistance. We are also indebted to BoehringerIngelheim for their generous gifts of clonidine HCI and 4-hydroxyclonidine HBr. References
i) z) 3) 4)
5) 6) 7) 8) 9) io) 11)
12) 13) 14)
15) 16)
HOEFKE, W. and KOBINGER, W., Arzneimittel-Forsch. 16. 1038-1056 (1966) DOLLERY, C.T., DAVIES, D.S., DRAFFAN, G.H., DARGIE, H.3., DEAN, C.R., REID, 3.L., CLARE, R.A. and MURRAY, S., Clin Pharmacol Ther., 1__9,11-17 (1976) DAS, S.M., AHUJA, G.N. and NAIRAINASWAMY, A.S., Acts Neurol. Stand., 60 (/4), 214-217 (1979) GOLD, M.S., POTTASH, A.C. and KLEBER, H.D., Lancet, 1 (8220), 621 (i98i) BJORKQVIST, S.E., Aeta Psychiat. Scand (Denmark), 52 (4), 256-263 (1975) PAALZOW, G and PAALZOW, L., Naunyn-Schmiedeb. Arch Pharmacol. 2_92, i19-126 (1976) FIELDING, S., WIKLER, J., HYNES, M., SZEWCZAK, M., NOVICK, W.J. and LAL, H., J. Pharm. Exp. Ther., 207 (3), 899-905 (1978) STOCKHAUS, K. From "Problems of Drug Dependence", Nat. Acad. Sol. 355-366, (1977) TYERS, M.B., Br. 3. Pharmac., 6_99,503-512 (1980) SKINGLE, M. and TYERS, M.B., Br. 3. Pharmacol, 713, 323-327 (1980) HAYES, A.G. and TYERS, M.B., Brain Res., 189, 561-564 (1980) DUBUISSON, D. and DENNIS, S.G., Pain, 4, 161-174, (1977) PAALZOW, L., 3. Phar, Pharmac, 2._66,361-363 (1974) SKINGLE, M., and TYERS, M.B. 3. PharmacoI Methods, _2, 71-80 (1979) BIRITTAIN, R.T., and HANDLEY, S.L. 3. Physiol., Lond., 392, 805-813 (i967) POPICK, F.R., Life Sci, 18, 197-204 (1976)
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17) 18) 19)
Clonfdlne Antfnociceptlon
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CAHUSAC, P., HAYES9 A.G., SKINGLE, M. and TYERS, M.B. Brit. 3. Pharmacol, 7_44,96)P (1981) SPAULDING, T.C., VENAFRO, 3.3., Ma., M.G. and FIELDING, S. Neuropharm, ~ 103-105 (1979) SINHA, 3.N., ATKINSON, 3.M. and SCHMITT, H. Eur. 3. Pharmacol, 24, 11)-119 (197))
20) 21) 22) 2))
BENTLEY, G.A., COPELAND, I.W., STARR~ 3. Olin. Exp. Pharmac. Physiol. _4, 405-419 (1977) BENTLEY, (3.A. and STARR, 3. Clin. ExP. Pharmacol. Physiol, 6, 702 (1979) DREW, G.M. (Unpublished) 3ONES, B.3. and ROBERTS, D.3. 3. Pharm. Pharmac., 20, 302-304 (1968)