Effects of tizanidine, a centrally acting muscle relaxant, on motor systems

Gen. Pharrnac. Vol. 17, No. 2, pp. 137 142, 1986

0306-3623/86 $3.00+0.00 Copyright ~;. 1986 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

EFFECTS OF TIZANIDINE, A CENTRALLY ACTING MUSCLE RELAXANT, ON MOTOR SYSTEMS H. ONO,* K. MATSUMOTO, K. KATO, F. KATO, M. MIYAMOTO, T. MORI, T. NAKAMURA, J. OKA and H. FUKUDA Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo ll3, Japan (Received 7 June 1985)

Abstraet--l. Effects of tizanidine were studied with special reference to the effect on motor systems. 2. The drug effectively reduced the intercollicular decerebrate rigidity and y-activity indirectly recorded from muscle spindle afferent discharges without showing the direct inhibitory effect on muscle spindles in rats. 3. The drug dose-dependently inhibited the phasic responses of a-rigidity in anemic decerebrate rats without showing marked inhibition of the tonic response. 4. Tizanidine effectively depressed the crossed extensor reflex in chicks and depressed mono- and polysynaptic reflex potentials in rats; dorsal root reflex was increased transiently. 5. Tizanidine had no effect on the neuromuscular junction in rats and [3H]diazepam binding in rat brain membrane. 6. It is suggested that the depression by tizanidine of 7-system and spinal reflexes contribute to muscle relaxation and anti-spastic effects and that mechanisms of action are different from those of other centrally acting muscle relaxants such as mephenesin and benzodiazepines.

lntercollicular decerebrate rigidity in rats

INTRODUCTION Tizanidine (5-chloro-4-(2-imidazolin-2yl-amino)-2,1, 3 - b e n z o t h i a d i a z o l e - H C i , DS 103-282) is a new centrally acting muscle relaxant which m a y have supraspinal site o f action (Sayers et al., 1980) and is m o r e effective than baclofen in reducing muscle tone in spastic patients (Hassen and McLellan, 1980). In the present study, to further characterize the p h a r m a c o logical properties, the effects o f tizanidine on m o t o r systems and [3H]diazepam binding were investigated.

MATERIALS AND METHODS Anemic decerebrate rigidity in rats

Male Wistar rats (Nippon Rat Co.) were anesthetized with ether and the common carotid arteries were ligated (Fukuda et al., 1974). After a trephine opening (dia 5 mm) had been made in the central part of the occipital bone, the basilar artery was cauterized using bipolar tweezer electrodes of a coagulator (Mizuhoika Tokyo, Micro ID) and then ether anesthesia was stopped. Marked extension of the forelimbs and rigidity of the neck occurred within 30 min after the operation and lasted for more than 2 hr. The rat was placed on its back and the forelimb tension was measured by a semi-isotonic transducer (Togari et al., 1978). The tonic tension of the forelimbs (about 20 g) was termed the tonic response. The hind limbs were fixed at the ankle and the feet were stretched mechanically (rostrally 5 mm, for 5 sec, once every 60 sec; see figures in Togari et al., 1978 or Ono, 1982). The mechanical stimulation augmented the forelimb tension and this increase was termed the phasic response. At the end of experiments, the rat was killed by ether inhalation and tension in the most relaxed status of the forelimbs was recorded. *Author to whom correspondence should be addressed. 137

Under ether anesthesia, the mid-brain was stereotaxically transected by a spatula between the inferior and superior colliculi (Fukuda et al., 1972). In the course of recovery from anesthesia, rigidity appeared in the trunk and hind limbs. The rat was placed on its side and EMG was recorded by a coaxial needle electrode inserted into the gastrocnemius muscle. EMG activities from the muscle were amplified with an amplifier (Nihonkohden, RB-621G). EMG spikes higher than an arbitrary threshold were transformed into square wave pulses and fed into an integrator (reset time, 2 sec), the output of which was recorded by a polygraph (Nihonkohden, RM-6000). Artificial respiration was performed during the experiment, since tizanidine (0.14).3 mg/kg, i.v.) produced marked depression of spontaneous respiration in these preparations.

Gamma-motor activities

Rats were anesthetized with ct-chloralose (25 mg/kg, i.p.) and urethane (I g/kg, i.p.). Laminectomy was performed in the lumbo-sacral region and the spinal cord was exposed. Ventral roots from L4 to L6 on the left were left intact and dorsal roots and other ventral roots below L3 were cut bilaterally. The exposed spinal cord was covered with liquid paraffin kept at 36°C. The left hind limb was denervated, except the nerve innervating the triceps surae muscle. The muscle was dissected free from surrounding tissue. Achilles tendon was cut, connected to a stretching device and stretched with ramp-and-hold stretch. Afferent discharges were led from filaments of L5 dorsal root by bipolar silver-silver chloride wire electrodes, amplified, displayed on an oscilloscope (Nihonkohden, VC-9), transformed into square wave pulses and fed into an integrator (time constant, 0.1 sec), the output of which was recorded using an ink-writing recorder (San-ei Instrument, Rectigraph 8S). Gamma activity was induced by pinching the left ear and was detected indirectly as the change in the rate of muscle spindle afferent discharges (Takeuchi et al., 1978).

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Crossed extensor reflex in chicks Male chicks (40-80g) anesthetized with ct-chloralose (50mg/kg, i.p.) were fixed on their backs and artificially respired through a cannula inserted into the trachea. The superficial fibular nerve of the left leg was exposed and stimulated (0.2 Hz, duration 1 msec, supramaximal, Nihonkohden, MSE-20) to induce the contralateral extensor reflex. Movement of the right leg following stimulation was recorded by means of an isotonic transducer (Nihonkohden, TD-111S and JD-IllS) and a recorder (Toa, FBR-252A). Drugs were administered intraperitoneally through the cannula. Spinal reflexes in rats Rats were anesthetized with ct-chloralose (25 mg/kg, i.p.) and urethane (1 g/kg, i.p.). Laminectomy was performed in the lumbo-sacral region. Ventral and dorsal roots of the segments L4 and L5 were isolated. A skin pouch was formed at the site of the dissection to cover the exposed tissues with liquid paraffin kept at 36°C. The dorsal root of L5 was placed on bipolar silver wire electrodes for stimulation (0.2 Hz, 0.05 msec, supramaximal, Nihonkohden MSE-3). The ipsilateral ventral root of L5 and the dorsal root of L4 were placed on bipolar silver wire electrodes for recording. Monosynaptic (MSR) and polysynaptic reflexes (PSR) and dorsal root~lorsal root reflexes (DRR) were evoked in the L5 ventral root and in the L4 dorsal root, respectively. These reflex potentials were amplified with an amplifier (Nihonkohden, ABV-9), displayed on an oscilloscope (Nihonkohden, VC-9) and were averaged 8 times by an averaging computer (Nihonkohden, ATAC-350). The analog output was recorded by an ink-writing recorder (San-Ei Instrument, Rectigraph-8).

Drugs Drugs used were baclofen (CIBA-Geigy), ct-chloralose (Wako), chlorpromazine HC1 (Rh6ne-Poulenc), diazepam (Rh6ne-Poulenc), lidocaine-HCl (lwaki), mephenesin (Sigma), tizanidine (DS103-282, Sandoz), tolperisone-HC1 (Nippon Kayaku), d-tubocurarine CI (Tokyo Kasei) and urethane (Wako). Drugs dissolved in saline were administered intravenously (femoral vein), intraperitoneally or intraduodenally through the cannula.

RESULTS

Effects on anemic decerebrate rigidity Tizanidine (0.1-1 mg/kg, i.v.) dose-dependently suppressed the phasic responses o f anemic decerebrate rigidity; 1 m g / k g abolished the responses for 3 0 m i n (Fig. I A). O n the o t h e r hand, the effects o f tizanidine on the tonic responses saturated at the dose o f 0.3 mg/kg; the maximal depression was a b o u t 25% (Fig. 1B). Ten m g / k g o f intraduodenally administered tizanidine caused similar magnitude o f effect on the phasic and tonic responses to that o f tizanidine (1 mg/kg, i.v.), although the time course was slower. T o l p e r i s o n e - H C l (20 mg/kg, i.v.) transiently reduced b o t h responses as already reported by Togari et al. (1978) (Fig. I f ) . A

1OO

Muscle afferent discharges Effects of tizanidine on resting afferent discharges were studied using a similar preparation used in the measurement of 7-activity. The ventral roots were cut below L3 to eliminate the motor innervation of the muscle used. The triceps surae muscle was isolated from the surrounding tissue and the Achilles tendon was severed and loaded with tension of 10 g. Afferent discharges were recorded from split dorsal root fibers and integrated (reset time 2 sec) (Fukuda et al., 1974). Neuromuscular junction Rats were anesthetized with ct-chloralose (25 mg/kg, i.p.) and urethane (1 g/kg, i.p.). The twitch tension of the gastrocnemius-soleus muscle in response to nerve stimulation was recorded by means of a transducer (Nihonkohden, SB-IT) secured by a string to the distal tendon. The distal stump of the severed tibial nerve was stimulated by supramaximal square wave pulses, 0.2 Hz, 0.05 msec in duration, from a stimulator (Nihonkohden, MSE-3). The output of the transducer was amplified and recorded using a polygraph (Nihonkohden, RM-25). Artificial respiration was maintained during experiments. Assay o f [3H]diazepam binding to rat brain membranes Crude synaptic membranes were prepared from the rat whole brain excluding cerebellum by the method of Zukin et al. (1974). Briefly, membrane pellets were obtained by homogenization and centrifugation (Goto et al., 1983). The membranes were incubated with [3H]diazepam (0.5nM, 85 Ci/mmol; Amersham Japan) in the presence or absence of test drugs. Incubation was carried out for 60 min at 0 ~ ° C and terminated by adding 4 ml of ice-cold 50 mM Tris-HCl (pH 7.4), passing the preparation through a Whatman GF/B filter and washing with 4 ml of the same buffer. Specific binding was defined as the difference between the total binding and non-specific binding in the presence of 3 x 10 -6 M diazepam.

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Fig. I. Effects of tizanidine and tolperisone-HCl on the phasic and tonic responses of rigid forelimbs in anemic decerebrate rats. A: Effects of tizanidine on the phasic response. B: Effects of tizanidine on tonic response. O; 0.1 mg/kg, i.v. (n =4), O; 0.3 mg/kg (n = 5), f-I; 1.0mg/kg (n = 5). C: Effects of tolperisone-HCl (20 mg/kg, i.v.) on phasic (/X) and tonic (A) responses (n = 5). Ordinates: relative tension of each response. Abscissae: time in min after the injection of drugs. Each point represents the mean + SEM.

Muscle relaxant action of tizanidine 100--.e-

(n = 2) and 0.1 mg/kg, i.v. (n = 2) reduced spontaneous and evoked y-activities indirectly recorded from muscle afferent discharges. Active control chlorpromazine-HCl (0.2 mg/kg, i.v.) caused prolonged depressant effects on the activities (Fig. 3C).

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Effects on crossed extensor reflexes Tizanidine (0.1 and 0.3mg/kg) depressed the crossed extensor reflexes after intraperitoneal administration (Fig. 4). Time course of tizanidine was shorter than those of mephenesin and tolperisone.

Effects on intercollicular decerebrate rigidity E M G activities of 50-150 Hz were recorded from the rigid gastrocnemius muscle. As shown in Fig. 2, tizanidine dose-dependently reduced the E M G activity. The duration of action was very short; recovery from the effect of 0.1 and 0.3mg/kg tizanidine occurred within 10-15 and 20 min, respectively.

Effects on spinal reflex potentials The effects of tizanidine on the spinal reflex potentials were assessed in intact spinal cord rats (Table 1). At the doses of 0.01~).3 mg/kg, i.v., tizanidine dosedependently reduced the amplitude of MSR but 1 and 5 mg/kg i.v. of tizanidine did not show any significant effect on the MSR. PSR was increased at first and then depressed at 5 min after administration of tizanidine (0.01-5 mg/kg, i.v.). On the other hand, D R R was potentiated by tizanidine (0.01~0.3 mg/kg, i.v.), but mean effects of 1 and 5 mg/kg were not apparent. The time course of 0.3 mg/kg of tizanidine is shown in Fig. 5 and at this dose typical effects of tizanidine are seen.

Effects on y-activity As shown in Fig. 3, tizanidine 0.03 mg/kg, i.v.

Effects on muscle afferent discharges Tizanidine (0.03, 0.1, 0.3 and

Fig. 2. Dose-response relationship of tizanidine on the

EMG activity in intercollicular decerebrate rats. Ordinate: relative frequencies of EMG discharges at 1-5 min after injection when the depression is maximum, with SEM indicated. Abscissa: dose of drug. O; saline, O; tizanidine.

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Tizanidine

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Fig. 3. Effects of tizanidine and chlorpromazine-HC1 on 7-activity. Upper and lower records show integrated frequency of muscle spindle discharges and displacement developed in the triceps aurae muscle during the stretch (5 ram), respectively. Horizontal bars represent time for pinna pinching. Facilitation of the static afferent discharges during pinna pinching reflects activation of 7-activity. Calibrations: 100 Hz and 5 sec.

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Fig. 5. Time courses of effects of tizanidine (0.3 mg/kg, i.v.) on MSR (©), PSR ( 0 ) and DRR (I-q) in rats. Ordinate: means of amplitudes of reflexes in percent of controls. Abscissa: time in min after the injection of drugs. Each point represents the mean + SEM (n = 4).

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Fig. 4. Effects of tizanidine, mephenesin and tolperisoneHCI on the crossed extensor reflex in chicks. Ordinates: relative amplitude of leg extension. Each point represents the mean + SEM (n = 4). Abscissae: time in min after the injection of drugs. A: F-l; saline, O; tizanidine 0.1 mg/kg, i.p., O; tizanidine 0.3 mg/kg, i.p. B: A; tolperisone-HCl 10mg/kg, i.p., A; mephenesin 10 mg/kg, i.p.

show any effects on muscle twitch evoked by nerve stimulation, d-Tubocurarine C1 (0.6mg/kg, i.p.) caused strong inhibition on the twitch response (Fig. 7). Effects on [3H]diazepam binding Tizanidine and tolperisone ( 1 0 - 7 - 1 0 - 4 M ) did not inhibit the binding of [3H]diazepam to rat brain membrane (Fig. 8). DISCUSSION

n = 2-4) did not show decreasing effects on frequency of afferent discharges from the triceps-surae muscle (Fig. 6). Local anesthetic lidocaine-HCI effectively reduced the afferent discharges. Effects on neuromuscular junction

Tizanidine (100mg/kg, intraduodenally) did not Table 1, Effects o f tizanidine on spinal reflex potentials in rats Tizanidine

Amplitude o f reflexes ( % )

(mg/kg, i.v.)

n

1-5 min

MSR

saline 0,01 0.03 0.1 0.3 1 5

6 3 3 3 4 3 4

101.0 90.8 86.6 76.4 69.0 94.6 102.5

+ ± + ± + + +

1.6 a 0.2** 4.3** 6,4** 8,7* 2,7 5,4

100.6 89.6 86.1 80.0 66.4 89.7 95.8

+ 3.0 _+ 3.4 + 5.8* + 4.3** _ 8.1"* 4- 2.8 ± 3.2

PSR

saline 0.01 0.03 0.1 0.3 I 5

6 3 3 3 4 3 4

107.1 102.8 98.9 101.9 97.3 88.0 86.8

+ + + + ± ± ±

8,4 7.8 9.2 21.6 13.9 11.1 4.7

114.1 87.6 92.0 89.7 81.0 78.8 81.6

_ + + ± ± ± ±

DRR

saline 0.01 0.03 0.1 0.3 I 5

6 3 3 3 4 3 4

100.5 108.8 123.0 147.4 199.1 115.3 98.2

1 min + + + + + ± ±

1.8 4.4 11.7 38.1 31.8" 14.2 7.3

5-15 min

8.3 7.7 14.7 16.6 8.8* 19.1 5.2*

1-5 min 104.2 99.3 113.6 122.2 174.2 122.5 93.7

+ ± + + + ± +

6.6 13.1 5.4 21.8 17.8"* 20.8 30.1

~Mean + S E M o f relative reflex amplitudes expressed as the percentage o f that before d r u g administration. * P < 0.05; * * P < 0.01: Significantly different f r o m the saline-treated g r o u p (two-tailed Student's t-test or Welch's procedure (1937) when the population variance was unequal).

Tizanidine produced the reduction in muscle activities in rat rigidity models without showing inhibitory effects on neuromuscular junction (Figs 1, 2, 7). These results show that muscle relaxant action of tizanidine is not due to the peripheral action but due to spinal or supraspinal action of the drug. Tizanidine effectively depressed E M G activity in intercollicular decerebrate rats in which 7-motoneurone is tonically activated (Fig. 2). These results were confirmed by recording of ?-activity from muscle afferent discharges (Fig. 3). Afferent discharges from de-efferented muscle were not inhibited by tizanidine (Fig. 6), although the afferent discharges have been shown to be depressed by mephenesin-type muscle relaxants which have local anesthetic-like membranestabilizing action (Ono et al., 1984). Muscle spindles together with their afferent fibers and ?-motoneurones constitute an important complex within the system for controlling motor activity and it is probable that spastic movements of muscle disorder are mediated by this physiological neuronal loop. Thus, muscle relaxant effect of tizanidine may be partly due to the reduction of 7-motoneurone activity. Tonic response of ~t-rigidity was resistant to tizanidine (Fig. IA). The tonic responses have been shown to be reduced by some mephenesin-type muscle relaxants but not by some benzodiazepines (Togari et al., 1978; Ono, 1982; Goto et al., 1983). Tizanidine preferentially depressed the phasic responses of or-rigidity induced by mechanical stimulation of hind limbs. The phasic responses have been shown to be selectively reduced by benzodiazepine-like drugs (Togari et al., 1978; Goto et al., 1983) which cause the enhancement of GABAergic presynaptic inhibition in the spinal cord (Stratten and Barnes, 1971; Polc et aL,

Muscle relaxant action of tizanidine

141

Tizonidine ( 0.1 rag/kg, i.v. )

Tizanidine (1 m g / k g , i.v. )

Lidocaine- HCI ( 2 0 m g / k g , i.v. )

Fig. 6. Effects of tizanidine and lidocaine-HC1 on afferent discharges from muscle spindles in triceps surae muscle of rats. The muscle was subjected to a tension of 10 g throughout the experiments. Calibrations: 100 Hz and 5 rain. 1974) and may be a type of animal model for exaggerated abnormal reflexes in spastic paresis (Togari et al., 1978). Tizanidine (0.1-5mg/kg, i.v.) decreased PSR potentials in anesthetized rats, but the magnitude of effect was the same level at all dose ranges 5-15 min after administration. Tizanidine (0.1 and 0.3 mg/kg, i.p.) depressed the crossed extensor reflex in chicks (Fig. 4). These effects of tizanidine were stronger than those of mephenesin-type drugs, benzodiazepine-like drugs and baciofen (Fig. 4A; Ono et al., 1984; Goto et al., 1983; Fukuda et al., 1977). These effects on PSR may have some relation to the reported strong depressant effect on firing of interneuron in spinal dorsal horn (Davies, 1982; Davies et al., 1984). On the MSR and DRR, tizanidine did not show linear dose-related effects. Tizanidine (0.01-0.3 mg/ kg, i.v.) dose-dependently depressed MSR and increased DRR, whereas i and 5 mg/kg of tizanidine produced weaker or no effect on the reflexes; marked

effects on MSR, PSR and DRR were obtained at the dose of 0.3 mg/kg, i.v. DRR has been shown to be enhanced by benzodiazepines and considered to be an index of state of presynaptic inhibition (Schlosser, 1971; Stratten and Barnes, 1971; Poic et al., 1974). These effects of tizanidine on MSR and DRR are different from the results of Sayers et al. (1980), in which no effect and weak depressant effects were seen on MSR and DRR in cats, respectively. These differences may be due to species difference or the differences of experimental conditions. Tizanidine mimicked benzodiazepines; both drugs increased DRR and decreased the phasic response of or-rigidity (Togari et al., 1978; Ono, 1982; Goto et aL, 1983). This relationship prompted us to test affinity of tizanidine to benzodiazepine receptors. However,

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Fig. 7. Effects of tizanidine and d-tubocurarine C1 of the twitch tension of the triceps surae muscle of rats. Each points represents mean + SEM (n = 4-5). Ordinate: twitch tension expressed as the percentage of that before drug administration. Abscissa: time in min after the injection of drugs. O; tizanidine 100 mg/kg, i.d., ©; saline I ml, i.d., A; d-tubocurarine CI 0.6 mg/kg, i.p.

I 7

I 6

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-log(M)

Fig. 8. Effects of tizanidine and tolperisone on specific [~H]diazepam binding to rat brain membranes. Ordinate: [3H]diazepam binding with drugs expressed as the percent of control. Abscissa: molar concentration of drugs. Each points represents the mean_ SEM (n = 4). The area surrounded by broken lines shows SEM of control binding. O; tizanidine, O; tolperisone.

142

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tizanidine did not displace [3H]diazepam from the receptors. Since tizanidine does not have affinity for acetylcholine, dopamine or ~j-adrenaline receptors (Sayers et al., 1980; Davies, 1984; Coward et al., 1984), it may be considered that tizanidine has selective affinity to ~t2-adrenoceptor (Davies, 1984; Coward et al., 1984). Although the site of action of tizanidine on spinal reflexes is not obvious from the present results, it is possible that stimulation of supraspinal ~2-receptor by tizanidine may reduce the spontaneous descending stimulatory influences on the spinal cord. These possibilities are under investigation using ct2-agonists and antagonists in intact and spinal cord preparations. In summary, tizanidine has been shown to be a strong and new kind of muscle relaxant in comparison with mephenesin-type, benzodiazepine-type drugs and baclofen. Depression by tizanidine of 7-systems, phasic responses in or-rigidity, and spinal reflexes may contribute to muscle relaxant and antispastic effects of the drug. Acknowledgements--This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. The authors are grateful to Sandoz Pharmaceuticals Ltd for the supply of tizanidine. REFERENCES

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