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The effect of dantrolene sodium on rat skeletal muscle in relation to the plasma concentration
European Journal of Pharmacology, 53 (1979) 335--342 © Elsevier]North-Holland Biomedical Press
335
THE EFFECT OF DANTROLENE SODIUM ON RAT SKELETAL MUSCLE IN RELATION TO THE PLASMA CONCENTRATION W I L L E M J. M E Y L E R *, I N E K E M O L S - T H U R K O W HARRY WESSELING *
*, A R N O L D
H.J. S C A F **, S J A M S H I A H S A R G O
* and
• Institute of Clinical Pharmacology, and ** Department of Pharmacology, State University Groningen,
Bloemsingel 1, 9713 BZ Groningen, The Netherlands Received 2 March 1978, revised MS received 5 September 1978, accepted 18 October 1978
W.J. M E Y L E R , I. M O L S - T H U R K O W , A.H.J. SCAF, S. S A R G O and H. W E S S E L I N G , The effect of dantrolene sodium on rat skeletal muscle in relation to the plasma concentration, European J. Pharmacol. 53 (1979) 335--
342. Dantrolene sodium is a muscle relaxant used in the treatment of spasticity. It has been shown to interfere with calcium release from the sarcoplasmic reticulum and thus to inhibit excitation--contraction coupling. The effect of dantrolene sodium on the twitch tension of the tibialis anterior muscle of the rat was measured after 2 mg/kg i.v. or 25 mg/kg orally. Plasma concentrations were estimated at maximum twitch depression and during recovery from the block. In a separate series of experiments the half-life of labelled dantrolene sodium was measured in blood plasma, skeletal muscle and heart muscle of rats. Dantrolene sodium 2 mg/kg i.v. gave a maximal block of approximately 47%, the mean dantrolene sodium concentration was then 5.8 pg/ml. A half-life for distribution of 1.1 rain and an elimination half-life of 31 rain after intravenous administration were observed, elimination rate constants in skeletal and heart muscle were comparable. Recovery from the block went much slower, the half-time of the process being approximately 80 rain. Dantrolene sodium 25 mg/kg orally gave a maximal block of approximately 38% at a mean plasma concentration of 3.6 pg/ml after 14 rain. The recovery was again very slow. These experiments demonstrated that dantrolene sodium acts according to a two-compartment pharmacokinetic model. There was a discrepancy between duration of effect and plasma concentration of dantrolene sodium in the rat. This suggests that the receptor for dantrolene sodium is not located in the central compartment. Dantrolene sodium
Pharmacokinetics
Rat
1. I n t r o d u c t i o n D a n t r o l e n e s o d i u m (1- { [ 5 - ( p - n i t r o p h e n y l f u r f u r y l i d e n e ] a m i n o } h y d a n t o i n s o d i u m ) is a m u s c l e r e l a x a n t w h i c h acts b y depressing t h e c o n t r a c t i l i t y o f skeletal muscle. T h e d r u g is used for the treatment of spasticity (Monster, 1 9 7 4 ; C h i p m a n et al., 1 9 7 4 ; L a d d et al., 1 9 7 3 ) . In a n i m a l e x p e r i m e n t s it is also effective against p r o v o k e d m a l i g n a n t h y p e r t h e r m i a ( H a r r i s o n , 1 9 7 5 ; J a r d o n et al., 1 9 7 6 ; A n d e r son a n d J o n e s , 1976). I t was s h o w n t o i n h i b i t e x c i t a t i o n - - c o n t r a c t i o n coupling, p o s s i b l y b y r e d u c t i o n o f t h e c a l c i u m e f f l u x f r o m t h e sarc o p l a s m a t i c r e t i c u l u m (Ellis a n d C a r p e n t e r
1 9 7 4 ; B r o c k l e h u r s t 1 9 7 5 ; H a i n a u t a n d Desmedt 1974; Desmedt and Hainaut, 1977; Morgan and Bryant, 1977). I t has n o e f f e c t o n n e u r o m u s c u l a r transm i s s i o n (Ellis a n d C a r p e n t i e r , 1 9 7 2 ) or electrical p r o p e r t i e s o f t h e p o s t s y n a p t i c m e m b r a n e (Ellis a n d B r y a n t , 1 9 7 2 ; N o t t a n d B o w m a n , 1974). M o r e o v e r d a n t r o l e n e s o d i u m m a y depress t h e f r e q u e n c y o f t h e s p o n t a n e o u s m i n i a t u r e p o t e n t i a l s o f t h e frog n e u r o m u s c u l a r junction (Statham and Duncan, 1976). In this s t u d y we i n v e s t i g a t e d t h e relationship b e t w e e n t h e m u s c l e r e l a x a n t e f f e c t o n skeletal m u s c l e a n d t h e p l a s m a a n d tissue concentrations of dantrolene sodium following
336
W.J. M E Y L E R E T AL.
intravenous (i.v.) and oral (p.o.) administration in the rat. Moreover we followed the disappearance of the drug from the plasma and tissue after i.v. injection.
stant twitch tension. In another series, dantrolene sodium 25 mg/kg was administered orally; the rats were sacrificed at the time when the maximal block was reached and blood was collected.
2. Materials and methods
2.3. Comparative pharmacokinetic ments in plasma and various tissues.
Male white Wistar rats of 200--300 g weight were used. All experiments in which muscle contractions were measured were carried out under pentobarbital anaesthesia. The rats were artificially ventilated with air. Blood pressure was monitored continuously with a catheter inserted in the external carotid artery. Rectal temperature was kept constant at 37--38°C using a heating lamp. Drugs were administered either i.v. by injection into the external jugular vein or p.o. via a tube into the stomach. Both i.v. and p.o. solutions contained 5 mg/ml dantrolene sodium dissolved in propylene glycol: distilled H20 9 : 1 at pH 12 (Nott and Bowman, 1974).
2.1. Dose--(maximal) response experiments Dantrolene sodium was administered i.v. as bolus injections of 1, 2 and 4 mg/kg. The isometric twitch tension of the left tibialis anterior muscle, elicited by indirect supramaximal square wave stimulation of 0.3 msec duration at a rate of 0.2 Hz by a Grass stimulator was continuously measured with a Hottinger--Baldwin force displacement transducer, amplified by a Hottinger amplifier and recorded with a Riker Denshi recorder. Since twitch depressions did not return to normal within hours, only one dose of dantrolene sodium was used in each experiment.
2.2. Plasma ments
concentrations--effect
experi-
In one series of experiments, where dantrolene sodium 2 mg/kg was injected, rats.were decapitated and blood samples were collected at the time of maximal twitch depression and at 60, 75 and 90% recovery to the initial con-
experi-
14C-dantrolene sodium, which was a gift from the Norwich Pharmaceutical Co. Norwich N.Y. was supplemented with cold dantrolene sodium and 2 mg/kg doses were administered into the tail-vein by bolus injection to conscious rats. The animals were decapitated at different time intervals, blood was collected and the heart and the tibialis anterior muscle were removed and washed for 15 min. in a cooled Krebs--Hepes solution to remove extracellular label. The tissues were homogenized in three parts of distilled water; 0.5 ml samples were dissolved in 15 ml Plasmasol scintillation fluid and counted for 10 min each in a Isocap-5 scintillation counter for total radioactivity. In a separate series, cold dantrolene sodium in the same doses as in the radioactive experiments (2 mg/kg) was administered by injection into the tail-vein and the rats were decapitated at various time intervals. Blood was collected and the concentrations of dantrolene sodium were estimated in plasma using a modification of the spectrofluorometric method described by Hollifield and Conklin (1973). The results are expressed as the mean + S.E.M.
3. Results
3.1. Dose--maximum effect relationship Dantrolene sodium gave a dose-dependent reduction of the twitch tension of the tibialis anterior muscle of the rat in vivo (fig. 1). The maximal block was achieved within 3--5 min after bolus injection. After 2 mg/kg, the maximal block was 46.9 + 1.3%. Recovery
DANTROLENE SODIUM CONCENTRATION--EFFECT RELATION
80. 70. 60, 2 0 ~ n=3
50. 40. 30. 20. 10,
mg/kg Fig. 1. Dose--response curve o f dantrolene sodium on the twitch tension of the rat tibialis anterior muscle. Each point represents m e a n _+ S.E.M. Ordinate: % decrease o f t w i t c h tension.
of the twitch tension to control values was very slow, both after i.v. and p.o. administration, the half-time of recovery (i.v.) being about 80 min.
3.2. Plasma concentration--effect relationship On average, a maximal block of 47% developed after 2 mg/kg dantrolene sodium i.v. A
337
mean plasma concentration of dantrolene of 5.8 #g/ml was then found. The plasma concentrations and times at various degrees of block are given in table 1. It can be derived from the data {table 1, last column) that the ratio between recovery of muscle contractions and the decline in log plasma concentrations was n o t constant during the time of the observations. This is shown graphically in fig. 2 where the individual plasma concentrations at four degrees of block are plotted versus time. If there were a direct relationship between plasma concentration and effect, one might expect that identical degrees of block should involve, with some random variation, approximately identical plasma concentrations. This is not so, however the plasma concentrations for identical degrees of block were correlated with time (P < 0.05 for 60, 75, 90%), thus indicating the existence of a barrier between locus of action and central compartment. After p.o. administration of 25 mg/kg dantrolene sodium, a maximum twitch depression of 38 + 1.8% developed after 14 + 1.7 min (n = 5). The corresponding mean plasma concentration was 3.6 + 0.4 p g / m l Though this corresponds with the i.v. data, the relations between duration of block and plasma concentrations may differ from those found after i.v. injection, since absorption from the G.I. tract is probably still in progress.
TABLE 1 Plasma c o n c e n t r a t i o n s of dantrolene s o d i u m at various degrees of block. Rates of decline of plasma c o n c e n t r a t i o n and recovery f r o m paralysis are c o m p a r e d in the last column. Degree of block at which rats (n) were sacrified Maximal
T i m e after injection (min +_ S.E.M.)
3.5 +- 0.35
Concentration of dantrolene s o d i u m (pg/ml +- S.E.M.) 5.8 +_ 0.4
(n = 4 )
60% recovery
0.028 18.3 +
2.3
3.7 + 0.5
81.2 -+ 16.8
1.2 +_ 0.2
104.8 +- 16.4
0.6 _+ 0.2
(n= 5) 75% recovery
0.031
(n= 5) 90% recovery
(n = 5)
% decrease in log plasma conc./% recovery
0.020
338
W.J. MEYLER ET AL.
pg/mt
3.3. The half life o f dantrolene sodium in blood and tissue
10.
1.0. x
x
\
0.1
2'0 ,;
6'0 8'0 1;o lio
lio
min. Fig. 2. Plasma concentrations corresponding with various degrees of block, plotted against time. • concentrations at maximal decrease of the twitch tension; • concentrations at 60% recovery; o concentrations at 75% recovery; • concentrations at 90% recovery.
Fig. 3 shows the decay curves of total radioactivity (i.e. dantrolene sodium plus eventual metabolites) in r~t plasma tibialis anterior muscle and heart muscle after i.v. administration. All three curves run parallel; the relevant pharmacokinetic data are given in table 2. The plasma curve can be described according to a two-compartment pharmacokinetic model and the figures suggest that both skeletal and heart muscle belong to the plasma i.e. central compartment. This is consistent with the rapid onset of action of dantrolene sodium in the tibialis anterior muscle, but not with the slow recovery which suggests that there might be a third, very small compartment, filling very rapidly but with a very slow release. Further, it can be seen that the concentration in plasma at any
TABLE 2 Pharmacokinetic parameters of dantrolene sodium in plasma and dantrolene sodium plus metabolites in plasma, skeletal muscle and heart muscle. Plasma Dantrolene sodium
A
(Ug/ml) (min-1 ) B (pg/ml) (rain -1 ) Vc (l/kg) Vd (l/kg) k12 (min -1) k21 (min -1) kl0 (min -1) Tl/2a (rain) T1/23 (rain) kel (1 min -1 kg -1 )
10.9 0.618 8.0 0.0225 0.105 0.24 0.3153 0.2746 0.0506 1.12 31 0.00531
Dantrolene sodium + metabolites
7.74 0.0180
38
Skeletal muscle
Heart
Dantrolene sodium + metabolites
Dantrolene sodium + metabolites
2.40 0.0206
34
10.46 0.508 4.15 0.0207
1.37 33.5
D A N T R O L E N E SODIUM C O N C E N T R A T I O N - - E F F E C T R E L A T I O N
I00.
10.
Fig. 3. C o n c e n t r a t i o n / t i m e c u r v e o f d a n t r o l e n e s o d i u m plus m e t a b o l i t e s in p l a s m a ($ . . . . . 4 ) , s k e l e t a l m u s c l e (A A) a n d h e a r t m u s c l e (o . . . . . . o) a f t e r 2 m g / k g l a b e l l e d d a n t r o l e n e s o d i u m i.v. in rats (n = 2).
o.. o . . . . . . . . . . b.
@
'dL
339
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i
o
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0.01
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2'O '
~'0 '
O~ '
Ob ' time
160 '
1~0
1~0 '
1~0 '
160
(mil~)
time is about four times that in skeletal muscle and only twice the concentration in heart muscle. The plasma concentration/time curve for dantrolene sodium plus its metabolites and the plasma concentration curve of dantrolene sodium alone are given in fig. 4. The difference between these t w o curves
represents the metabolites, probably for a major part the assumed 5-OH derivative, which is the major active metabolite (Ellis and Wessels, 1978). Since 5-OH dantrolene is not available in labelled form, exact concentrations could not be determined. Both curves run roughly parallel and there are no great
100.
Fig. 4. C o n c e n t r a t i o n / t i m e curve of dantrolene sodiu m (+ . . . . . . + ) a n d d a n t r o l e n e s o d i u m plus its m e t a b o l i t e s (= ~ ) in p l a s m a after 2 m g / k g l a b e l l e d d a n t r o l e n e s o d i u m i.v. in rats (n = 2).
10
(11.
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2~0
'
/~0
'
f:~
'
I10
' time
100 (rain)
'
120
'
1~*0
'
160
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100
340
differences between the concentrations. In view of this it is acceptable to use the combined curve in relation to the muscle relaxant effect. The apparent volume of distribution (VD) of dantrolene sodium was estimated with the formula VD = D • ~/(A~ + B~) (the parameters A, B, ~ and {3 were obtained by iterative peeling). VD was relatively small (24%) probably representing the extracellular water. The maximal block of 47% decreased by 50% in about 80 min.
4. Discussion The conc entration--time relationship ( fig. 2) and the disproportionality between the log plasma concentrations and the recovery (table 2) indicate that the site of action of dantrolene sodium is not in the central i.e. plasma compartment. This is n o t surprising, since it is reported that the drug acts intracellularly (Ellis and Carpenter, 1974). Since the semi-log plasma concentrations and muscle decay curve were parallel, this discrepancy between plasma concentrations and effect also holds true for the peripheral compartment. The discrepancy may be due to several causes. First it is possible that dantrolene sodium does not follow a linear relationship between log concentration at the receptor site and effect. It is unlikely that during the fi-phase in our experiments an overdosage was present, since we found a linear dose--response curve (fig. 1) in vivo and the concentrations used gave approximately 47% block. Secondly, dantrolene sodium may remain much longer at the receptor site than in the surrounding intracellular, non reactive space. However, a third compartment representing these receptor sites (i.e. Ca 2÷ binding places in the sarcoplasmatic reticulum) is very small in relation to the intracellular space and thus pharmacokinetic details concerning the "effective" compartment will be completely obscured during the fi-phase and be below detection level later on. Detailed investigations on
W.J. MEYLER ET AL.
the binding affinity of isolated sarcoplasmatic reticulum might throw more light on this problem. Finally, an active metabolite might interfere and be responsible for the differences between effect and tissue concentration. Dantrolene sodium is metabolised in the liver mainly by 5-hydroxylation of the hydantoin group and for a small part by reduction of the nitro group followed by acetylation (Hollifield and Conklin, 1973; Lietman et al., 1974). Even if the major metabolite, 5-hydroxydantrolene, and the parent drug were equally potent, the differences between half lives of metabolite and parent drug are t o o small to explain these discrepancies. Thus the second explanation, intensive binding to the receptor site is the most likely one. The equilibrium between plasma and biophase was only reached in a later state which may indicate that the estimation of plasma concentrations of dantrolene sodium in relation to effect is only meaningful under steady state conditions. After oral administration to rats we found a peak effect of a huge dose (25 mg/ kg). Plasma concentrations were comparable with those after i.v. injection of approximately equipotent doses. Herman et al. (1972) found peak effects of 40--70% in patients 4 h after oral ingestion of about 2 mg/kg dantrolene sodium, and concomitant plasmaconcentrations of 1.25 pg/ml. Monster et al. (1973) observed plasma concentrations of 0.7--1.45 pg/ml, 3--6 h after oral administration of 100--125 mg dantrolene sodium (about 1.5 mg/kg). Species-dependent rate constant for absorption and distribution into muscle and re-entry into the central compartment most probably caused the differences with our experiments. In a previous study (Meyler et al. 1976) we found that dantrolene sodium also affected the contractility of the spontaneously beating rat heart in vitro; this finding was confirmed by Bowman and Khan (1977) in studies on isolated atria, although with higher concentrations than used on the rat diaphragm. This is in contrast with others
DANTROLENE SODIUM CONCENTRATION--EFFECT RELATION
(Ellis et al., 1973, 1976; Butterfield and Ellis, 1973; Ellis et al., 1975 and Harrison, 1975), who found no significant changes in myocardial contractility in different species in vivo. One explanation of these differences might be that, in vivo, the effect of dantrolene sodium on cardiac parameters is masked by compensating mechanisms of the cardiovascular system. In in vitro experiments, Van Winkle (1976) also observed no effects on contractility of myocardial fibers, whereas Ellis (1976), using atrial strips, obtained an effect on contractility, however, at much higher concentrations of dantrolene sodium than necessary for skeletal muscle relaxation. The observations in vitro are only relevant if the concentrations are comparable with in vivo situations. We found a 50% decrease of contractility of cardial muscle in vitro at a concentration of 8/~g/ml dantrolene sodium. In vivo with a muscle relaxant effect of dantrolene sodium comparable with clinical situations (Herman, 1972) we found peak concentrations of approximately 6 pg/g tissue in heart muscle of the rat. Though it is difficult to extrapolate these data to human stituations, this might eventually be important if patients with malignant hyperthermia were to be treated with high doses of dantrolene sodium intravenously. In conclusion we may say that the pharmacokinetic behaviour of dantrolene sodium in rats offers an explanation for the long lasting effects of the drug. Though the complex of absorption, distribution and excretion kinetics in man will probably differ from that in rats, it may be expected that in man also it is only steady state plasma concentrations which will correlate with effect. The study of these concentrations, reached after prolonged oral medication, in relation to effect is in progress in our laboratory.
Acknowledgements We wish to thank Dr. S. Agoston for criticism. We thank Mr. M.C. Houwertjes and Mr. E. Scholtens for
341
technical and Mrs. U.W. Kersten-Kleef for analytical assistance.
References Anderson, I.L. and E.W. Jones, 1976, Porcine malignant hyperthermia: the effect of dantrolene sodium on in vitro induced contraction of susceptible muscle, Anaesthesiology 44, 57. Brocklehurst, L., 1975, Dantrolene sodium and "skinned" muscle fibers, Nature (London) 254, 364. Bowman, W.C. and H.H. Khan, 1977, Effects of dantrolene sodium on isolated skeletal smooth and cardiac muscle of the guinea pig, J. Pharm. Pharmacol. 29, 628. Butterfield, J.L. and K.O. Ellis, 1973, Effects of dantrolene sodium, a skeletal muscle relaxant, on the contractility of cardiac and smooth muscle, Federation Proc. 32, 772. Chipman, M., S. Kaul and M. Lambie, 1974, Efficacy of dantrolene sodium in the treatment of spasticity, Dis. Nerv. Syst. 35,427. Desmedt, J.E. and K. Hainaut, 1977, Inhibition of the intracellular release of calcium by dantrolene in barnacle giant muscle fibers, J. Physiol. 265, 565. Ellis, K.O. and S.H. Bryant, 1972, Excitation--contraction uncoupling in skeletal muscle by dantrolene sodium, Naunyn-Schmiedeb. Arch. Pharmacol. 274, 107. Ellis, K.O. and J.E. Carpenter, 1972, Studies on the mechanism of action of dantrolene sodium a skeletal muscle relaxant, Naunyn-Schmiedeb. Arch. Pharmacol. 275, 83. Ellis, K.O. and J.F. Carperter, 1974, Mechanism of control of skeletal muscle contraction by dantrolene sodium, Arch. Phys. Med. Rehabil. 55, 362. Ellis, K.O., A.W. Castellion, C.J. Honkomp, F.L. Wessels, J.F. Carpenter and R.P. Halliday, 1973, Dantrolene, a direct acting skeletal muscle relaxant, J. Pharm. Sci. 62, 948. Ellis, K.O., J.L. Butterfield, F.L. Wessels and J.F. Carpenter, 1976, A comparison of skeletal, cardiac and smooth muscle actions of dantrolene sodium - - a skeletal muscle relaxant, Arch. Intern. Pharmacodyn. Therap. 244, 1. Ellis, K.O. and F.L. Wessels, 1978, Pharmacological studies of the identified metabolites of dantrolene sodium, Naunyn-Schmiedeb. Arch. Pharmacol. 301,237. Ellis, R.H., P. Simpson, P. Tatham, M. Leighton and J. Williams, 1975, The cardiovascular effect of dantrolene sodium in dogs, Anaesthesia 30, 318. Hainaut, K. and J.E. Desmedt, 1974, Effect of d a n -
342 trolene sodium on calcium movements in single muscle fibers, Nature 252, 5485, p. 728. Harrison, G.G., 1975, Control of the malignant hyperpyrexic syndrome in MHS swine by dantrolene sodium, Brit. J. Anaesth. 47, 62. Herman, R., N. Mayer and S.A. Mecomber, 1972, Clinical pharmaco-physiology of dantrolene sodium, Amer. J. Phys. Med. 51, 6. Hollifield, R.D. and J.D. Conklin, 1973, Determination of dantrolene in biological specimens containing drug-related metabolites, J. Pharm. Sci. 62, 271. Jardon, O.M., D. Kerr, R. Cochram and D.W. Wingard, 1976, Malignant hyperthermia -- a disease of importance to orthopedics (study of dantrolene in porcine malignant hyperthermia), J. Bone Joint Surg. 58, 738. Ladd, H.W., G. Gingras, J. Boucher, N. Kazdan and R. Keenan, 1973, The reductive effect of a new pharmacological agent (dantrolene sodium) on spasticity, Union Med. Can. 102, 1087. Lietman, P.S., R.H.A. Haslam and J.R. Walcher, 1974, Parmacology of dantrolene sodium in children, Arch. Phys. Med. Rehahil. 55, 388.
W.J. MEYLER ET AL. Meyler, W.J., H. Weaseling and S. Agoston, 1976, The effets of dantrolene sodium on cardiac and skeletal muscle in rats, European J. Pharmacol. 39, 127. Monster, A.W., R. Herman, S. Meeks and J. McHenry, 1973, Cooperative study for assessing the effects of a pharmacological agent on spasticity, Amer. J. Phys. Med. 52, 4. Monster, A.W., 1974, Spasticity and the effect of dantrolene sodium, Arch. Phys. Med. Rehab. 55, 373. Morgan, K. and S.H. Bryant, 1977, The mechanism of action of dantrolene sodium, J. Pharmacol. Exptl. Therap. 201, 1. Nott, M.W. and W.C. Bowman, 1974, Actions of dantrolene sodium on contractions of the tibialis anterior and soleus muscles of cats under chloralose anaesthesia, Clin. Exptl. Pharmacol. Physiol. 1,113. Statham, H.E. and C.J. Duncan, 1976, Dantrolene and the neuromuscular junction: Evidence for intracellular calcium stores, European J. Pharmacol. 39, 143. Van Winkle, W.B., 1976, Calcium release from skeletal muscle sarcoplasmatic reticulum: site of action of dantrolene sodium, Science 193, 1130.
335
THE EFFECT OF DANTROLENE SODIUM ON RAT SKELETAL MUSCLE IN RELATION TO THE PLASMA CONCENTRATION W I L L E M J. M E Y L E R *, I N E K E M O L S - T H U R K O W HARRY WESSELING *
*, A R N O L D
H.J. S C A F **, S J A M S H I A H S A R G O
* and
• Institute of Clinical Pharmacology, and ** Department of Pharmacology, State University Groningen,
Bloemsingel 1, 9713 BZ Groningen, The Netherlands Received 2 March 1978, revised MS received 5 September 1978, accepted 18 October 1978
W.J. M E Y L E R , I. M O L S - T H U R K O W , A.H.J. SCAF, S. S A R G O and H. W E S S E L I N G , The effect of dantrolene sodium on rat skeletal muscle in relation to the plasma concentration, European J. Pharmacol. 53 (1979) 335--
342. Dantrolene sodium is a muscle relaxant used in the treatment of spasticity. It has been shown to interfere with calcium release from the sarcoplasmic reticulum and thus to inhibit excitation--contraction coupling. The effect of dantrolene sodium on the twitch tension of the tibialis anterior muscle of the rat was measured after 2 mg/kg i.v. or 25 mg/kg orally. Plasma concentrations were estimated at maximum twitch depression and during recovery from the block. In a separate series of experiments the half-life of labelled dantrolene sodium was measured in blood plasma, skeletal muscle and heart muscle of rats. Dantrolene sodium 2 mg/kg i.v. gave a maximal block of approximately 47%, the mean dantrolene sodium concentration was then 5.8 pg/ml. A half-life for distribution of 1.1 rain and an elimination half-life of 31 rain after intravenous administration were observed, elimination rate constants in skeletal and heart muscle were comparable. Recovery from the block went much slower, the half-time of the process being approximately 80 rain. Dantrolene sodium 25 mg/kg orally gave a maximal block of approximately 38% at a mean plasma concentration of 3.6 pg/ml after 14 rain. The recovery was again very slow. These experiments demonstrated that dantrolene sodium acts according to a two-compartment pharmacokinetic model. There was a discrepancy between duration of effect and plasma concentration of dantrolene sodium in the rat. This suggests that the receptor for dantrolene sodium is not located in the central compartment. Dantrolene sodium
Pharmacokinetics
Rat
1. I n t r o d u c t i o n D a n t r o l e n e s o d i u m (1- { [ 5 - ( p - n i t r o p h e n y l f u r f u r y l i d e n e ] a m i n o } h y d a n t o i n s o d i u m ) is a m u s c l e r e l a x a n t w h i c h acts b y depressing t h e c o n t r a c t i l i t y o f skeletal muscle. T h e d r u g is used for the treatment of spasticity (Monster, 1 9 7 4 ; C h i p m a n et al., 1 9 7 4 ; L a d d et al., 1 9 7 3 ) . In a n i m a l e x p e r i m e n t s it is also effective against p r o v o k e d m a l i g n a n t h y p e r t h e r m i a ( H a r r i s o n , 1 9 7 5 ; J a r d o n et al., 1 9 7 6 ; A n d e r son a n d J o n e s , 1976). I t was s h o w n t o i n h i b i t e x c i t a t i o n - - c o n t r a c t i o n coupling, p o s s i b l y b y r e d u c t i o n o f t h e c a l c i u m e f f l u x f r o m t h e sarc o p l a s m a t i c r e t i c u l u m (Ellis a n d C a r p e n t e r
1 9 7 4 ; B r o c k l e h u r s t 1 9 7 5 ; H a i n a u t a n d Desmedt 1974; Desmedt and Hainaut, 1977; Morgan and Bryant, 1977). I t has n o e f f e c t o n n e u r o m u s c u l a r transm i s s i o n (Ellis a n d C a r p e n t i e r , 1 9 7 2 ) or electrical p r o p e r t i e s o f t h e p o s t s y n a p t i c m e m b r a n e (Ellis a n d B r y a n t , 1 9 7 2 ; N o t t a n d B o w m a n , 1974). M o r e o v e r d a n t r o l e n e s o d i u m m a y depress t h e f r e q u e n c y o f t h e s p o n t a n e o u s m i n i a t u r e p o t e n t i a l s o f t h e frog n e u r o m u s c u l a r junction (Statham and Duncan, 1976). In this s t u d y we i n v e s t i g a t e d t h e relationship b e t w e e n t h e m u s c l e r e l a x a n t e f f e c t o n skeletal m u s c l e a n d t h e p l a s m a a n d tissue concentrations of dantrolene sodium following
336
W.J. M E Y L E R E T AL.
intravenous (i.v.) and oral (p.o.) administration in the rat. Moreover we followed the disappearance of the drug from the plasma and tissue after i.v. injection.
stant twitch tension. In another series, dantrolene sodium 25 mg/kg was administered orally; the rats were sacrificed at the time when the maximal block was reached and blood was collected.
2. Materials and methods
2.3. Comparative pharmacokinetic ments in plasma and various tissues.
Male white Wistar rats of 200--300 g weight were used. All experiments in which muscle contractions were measured were carried out under pentobarbital anaesthesia. The rats were artificially ventilated with air. Blood pressure was monitored continuously with a catheter inserted in the external carotid artery. Rectal temperature was kept constant at 37--38°C using a heating lamp. Drugs were administered either i.v. by injection into the external jugular vein or p.o. via a tube into the stomach. Both i.v. and p.o. solutions contained 5 mg/ml dantrolene sodium dissolved in propylene glycol: distilled H20 9 : 1 at pH 12 (Nott and Bowman, 1974).
2.1. Dose--(maximal) response experiments Dantrolene sodium was administered i.v. as bolus injections of 1, 2 and 4 mg/kg. The isometric twitch tension of the left tibialis anterior muscle, elicited by indirect supramaximal square wave stimulation of 0.3 msec duration at a rate of 0.2 Hz by a Grass stimulator was continuously measured with a Hottinger--Baldwin force displacement transducer, amplified by a Hottinger amplifier and recorded with a Riker Denshi recorder. Since twitch depressions did not return to normal within hours, only one dose of dantrolene sodium was used in each experiment.
2.2. Plasma ments
concentrations--effect
experi-
In one series of experiments, where dantrolene sodium 2 mg/kg was injected, rats.were decapitated and blood samples were collected at the time of maximal twitch depression and at 60, 75 and 90% recovery to the initial con-
experi-
14C-dantrolene sodium, which was a gift from the Norwich Pharmaceutical Co. Norwich N.Y. was supplemented with cold dantrolene sodium and 2 mg/kg doses were administered into the tail-vein by bolus injection to conscious rats. The animals were decapitated at different time intervals, blood was collected and the heart and the tibialis anterior muscle were removed and washed for 15 min. in a cooled Krebs--Hepes solution to remove extracellular label. The tissues were homogenized in three parts of distilled water; 0.5 ml samples were dissolved in 15 ml Plasmasol scintillation fluid and counted for 10 min each in a Isocap-5 scintillation counter for total radioactivity. In a separate series, cold dantrolene sodium in the same doses as in the radioactive experiments (2 mg/kg) was administered by injection into the tail-vein and the rats were decapitated at various time intervals. Blood was collected and the concentrations of dantrolene sodium were estimated in plasma using a modification of the spectrofluorometric method described by Hollifield and Conklin (1973). The results are expressed as the mean + S.E.M.
3. Results
3.1. Dose--maximum effect relationship Dantrolene sodium gave a dose-dependent reduction of the twitch tension of the tibialis anterior muscle of the rat in vivo (fig. 1). The maximal block was achieved within 3--5 min after bolus injection. After 2 mg/kg, the maximal block was 46.9 + 1.3%. Recovery
DANTROLENE SODIUM CONCENTRATION--EFFECT RELATION
80. 70. 60, 2 0 ~ n=3
50. 40. 30. 20. 10,
mg/kg Fig. 1. Dose--response curve o f dantrolene sodium on the twitch tension of the rat tibialis anterior muscle. Each point represents m e a n _+ S.E.M. Ordinate: % decrease o f t w i t c h tension.
of the twitch tension to control values was very slow, both after i.v. and p.o. administration, the half-time of recovery (i.v.) being about 80 min.
3.2. Plasma concentration--effect relationship On average, a maximal block of 47% developed after 2 mg/kg dantrolene sodium i.v. A
337
mean plasma concentration of dantrolene of 5.8 #g/ml was then found. The plasma concentrations and times at various degrees of block are given in table 1. It can be derived from the data {table 1, last column) that the ratio between recovery of muscle contractions and the decline in log plasma concentrations was n o t constant during the time of the observations. This is shown graphically in fig. 2 where the individual plasma concentrations at four degrees of block are plotted versus time. If there were a direct relationship between plasma concentration and effect, one might expect that identical degrees of block should involve, with some random variation, approximately identical plasma concentrations. This is not so, however the plasma concentrations for identical degrees of block were correlated with time (P < 0.05 for 60, 75, 90%), thus indicating the existence of a barrier between locus of action and central compartment. After p.o. administration of 25 mg/kg dantrolene sodium, a maximum twitch depression of 38 + 1.8% developed after 14 + 1.7 min (n = 5). The corresponding mean plasma concentration was 3.6 + 0.4 p g / m l Though this corresponds with the i.v. data, the relations between duration of block and plasma concentrations may differ from those found after i.v. injection, since absorption from the G.I. tract is probably still in progress.
TABLE 1 Plasma c o n c e n t r a t i o n s of dantrolene s o d i u m at various degrees of block. Rates of decline of plasma c o n c e n t r a t i o n and recovery f r o m paralysis are c o m p a r e d in the last column. Degree of block at which rats (n) were sacrified Maximal
T i m e after injection (min +_ S.E.M.)
3.5 +- 0.35
Concentration of dantrolene s o d i u m (pg/ml +- S.E.M.) 5.8 +_ 0.4
(n = 4 )
60% recovery
0.028 18.3 +
2.3
3.7 + 0.5
81.2 -+ 16.8
1.2 +_ 0.2
104.8 +- 16.4
0.6 _+ 0.2
(n= 5) 75% recovery
0.031
(n= 5) 90% recovery
(n = 5)
% decrease in log plasma conc./% recovery
0.020
338
W.J. MEYLER ET AL.
pg/mt
3.3. The half life o f dantrolene sodium in blood and tissue
10.
1.0. x
x
\
0.1
2'0 ,;
6'0 8'0 1;o lio
lio
min. Fig. 2. Plasma concentrations corresponding with various degrees of block, plotted against time. • concentrations at maximal decrease of the twitch tension; • concentrations at 60% recovery; o concentrations at 75% recovery; • concentrations at 90% recovery.
Fig. 3 shows the decay curves of total radioactivity (i.e. dantrolene sodium plus eventual metabolites) in r~t plasma tibialis anterior muscle and heart muscle after i.v. administration. All three curves run parallel; the relevant pharmacokinetic data are given in table 2. The plasma curve can be described according to a two-compartment pharmacokinetic model and the figures suggest that both skeletal and heart muscle belong to the plasma i.e. central compartment. This is consistent with the rapid onset of action of dantrolene sodium in the tibialis anterior muscle, but not with the slow recovery which suggests that there might be a third, very small compartment, filling very rapidly but with a very slow release. Further, it can be seen that the concentration in plasma at any
TABLE 2 Pharmacokinetic parameters of dantrolene sodium in plasma and dantrolene sodium plus metabolites in plasma, skeletal muscle and heart muscle. Plasma Dantrolene sodium
A
(Ug/ml) (min-1 ) B (pg/ml) (rain -1 ) Vc (l/kg) Vd (l/kg) k12 (min -1) k21 (min -1) kl0 (min -1) Tl/2a (rain) T1/23 (rain) kel (1 min -1 kg -1 )
10.9 0.618 8.0 0.0225 0.105 0.24 0.3153 0.2746 0.0506 1.12 31 0.00531
Dantrolene sodium + metabolites
7.74 0.0180
38
Skeletal muscle
Heart
Dantrolene sodium + metabolites
Dantrolene sodium + metabolites
2.40 0.0206
34
10.46 0.508 4.15 0.0207
1.37 33.5
D A N T R O L E N E SODIUM C O N C E N T R A T I O N - - E F F E C T R E L A T I O N
I00.
10.
Fig. 3. C o n c e n t r a t i o n / t i m e c u r v e o f d a n t r o l e n e s o d i u m plus m e t a b o l i t e s in p l a s m a ($ . . . . . 4 ) , s k e l e t a l m u s c l e (A A) a n d h e a r t m u s c l e (o . . . . . . o) a f t e r 2 m g / k g l a b e l l e d d a n t r o l e n e s o d i u m i.v. in rats (n = 2).
o.. o . . . . . . . . . . b.
@
'dL
339
"-.o
"o.
" e.
•
-.
-..o
•
i
o
"-..
, t
', ,, ,, ',
0.01
'
2'O '
~'0 '
O~ '
Ob ' time
160 '
1~0
1~0 '
1~0 '
160
(mil~)
time is about four times that in skeletal muscle and only twice the concentration in heart muscle. The plasma concentration/time curve for dantrolene sodium plus its metabolites and the plasma concentration curve of dantrolene sodium alone are given in fig. 4. The difference between these t w o curves
represents the metabolites, probably for a major part the assumed 5-OH derivative, which is the major active metabolite (Ellis and Wessels, 1978). Since 5-OH dantrolene is not available in labelled form, exact concentrations could not be determined. Both curves run roughly parallel and there are no great
100.
Fig. 4. C o n c e n t r a t i o n / t i m e curve of dantrolene sodiu m (+ . . . . . . + ) a n d d a n t r o l e n e s o d i u m plus its m e t a b o l i t e s (= ~ ) in p l a s m a after 2 m g / k g l a b e l l e d d a n t r o l e n e s o d i u m i.v. in rats (n = 2).
10
(11.
001 '
2~0
'
/~0
'
f:~
'
I10
' time
100 (rain)
'
120
'
1~*0
'
160
'
100
340
differences between the concentrations. In view of this it is acceptable to use the combined curve in relation to the muscle relaxant effect. The apparent volume of distribution (VD) of dantrolene sodium was estimated with the formula VD = D • ~/(A~ + B~) (the parameters A, B, ~ and {3 were obtained by iterative peeling). VD was relatively small (24%) probably representing the extracellular water. The maximal block of 47% decreased by 50% in about 80 min.
4. Discussion The conc entration--time relationship ( fig. 2) and the disproportionality between the log plasma concentrations and the recovery (table 2) indicate that the site of action of dantrolene sodium is not in the central i.e. plasma compartment. This is n o t surprising, since it is reported that the drug acts intracellularly (Ellis and Carpenter, 1974). Since the semi-log plasma concentrations and muscle decay curve were parallel, this discrepancy between plasma concentrations and effect also holds true for the peripheral compartment. The discrepancy may be due to several causes. First it is possible that dantrolene sodium does not follow a linear relationship between log concentration at the receptor site and effect. It is unlikely that during the fi-phase in our experiments an overdosage was present, since we found a linear dose--response curve (fig. 1) in vivo and the concentrations used gave approximately 47% block. Secondly, dantrolene sodium may remain much longer at the receptor site than in the surrounding intracellular, non reactive space. However, a third compartment representing these receptor sites (i.e. Ca 2÷ binding places in the sarcoplasmatic reticulum) is very small in relation to the intracellular space and thus pharmacokinetic details concerning the "effective" compartment will be completely obscured during the fi-phase and be below detection level later on. Detailed investigations on
W.J. MEYLER ET AL.
the binding affinity of isolated sarcoplasmatic reticulum might throw more light on this problem. Finally, an active metabolite might interfere and be responsible for the differences between effect and tissue concentration. Dantrolene sodium is metabolised in the liver mainly by 5-hydroxylation of the hydantoin group and for a small part by reduction of the nitro group followed by acetylation (Hollifield and Conklin, 1973; Lietman et al., 1974). Even if the major metabolite, 5-hydroxydantrolene, and the parent drug were equally potent, the differences between half lives of metabolite and parent drug are t o o small to explain these discrepancies. Thus the second explanation, intensive binding to the receptor site is the most likely one. The equilibrium between plasma and biophase was only reached in a later state which may indicate that the estimation of plasma concentrations of dantrolene sodium in relation to effect is only meaningful under steady state conditions. After oral administration to rats we found a peak effect of a huge dose (25 mg/ kg). Plasma concentrations were comparable with those after i.v. injection of approximately equipotent doses. Herman et al. (1972) found peak effects of 40--70% in patients 4 h after oral ingestion of about 2 mg/kg dantrolene sodium, and concomitant plasmaconcentrations of 1.25 pg/ml. Monster et al. (1973) observed plasma concentrations of 0.7--1.45 pg/ml, 3--6 h after oral administration of 100--125 mg dantrolene sodium (about 1.5 mg/kg). Species-dependent rate constant for absorption and distribution into muscle and re-entry into the central compartment most probably caused the differences with our experiments. In a previous study (Meyler et al. 1976) we found that dantrolene sodium also affected the contractility of the spontaneously beating rat heart in vitro; this finding was confirmed by Bowman and Khan (1977) in studies on isolated atria, although with higher concentrations than used on the rat diaphragm. This is in contrast with others
DANTROLENE SODIUM CONCENTRATION--EFFECT RELATION
(Ellis et al., 1973, 1976; Butterfield and Ellis, 1973; Ellis et al., 1975 and Harrison, 1975), who found no significant changes in myocardial contractility in different species in vivo. One explanation of these differences might be that, in vivo, the effect of dantrolene sodium on cardiac parameters is masked by compensating mechanisms of the cardiovascular system. In in vitro experiments, Van Winkle (1976) also observed no effects on contractility of myocardial fibers, whereas Ellis (1976), using atrial strips, obtained an effect on contractility, however, at much higher concentrations of dantrolene sodium than necessary for skeletal muscle relaxation. The observations in vitro are only relevant if the concentrations are comparable with in vivo situations. We found a 50% decrease of contractility of cardial muscle in vitro at a concentration of 8/~g/ml dantrolene sodium. In vivo with a muscle relaxant effect of dantrolene sodium comparable with clinical situations (Herman, 1972) we found peak concentrations of approximately 6 pg/g tissue in heart muscle of the rat. Though it is difficult to extrapolate these data to human stituations, this might eventually be important if patients with malignant hyperthermia were to be treated with high doses of dantrolene sodium intravenously. In conclusion we may say that the pharmacokinetic behaviour of dantrolene sodium in rats offers an explanation for the long lasting effects of the drug. Though the complex of absorption, distribution and excretion kinetics in man will probably differ from that in rats, it may be expected that in man also it is only steady state plasma concentrations which will correlate with effect. The study of these concentrations, reached after prolonged oral medication, in relation to effect is in progress in our laboratory.
Acknowledgements We wish to thank Dr. S. Agoston for criticism. We thank Mr. M.C. Houwertjes and Mr. E. Scholtens for
341
technical and Mrs. U.W. Kersten-Kleef for analytical assistance.
References Anderson, I.L. and E.W. Jones, 1976, Porcine malignant hyperthermia: the effect of dantrolene sodium on in vitro induced contraction of susceptible muscle, Anaesthesiology 44, 57. Brocklehurst, L., 1975, Dantrolene sodium and "skinned" muscle fibers, Nature (London) 254, 364. Bowman, W.C. and H.H. Khan, 1977, Effects of dantrolene sodium on isolated skeletal smooth and cardiac muscle of the guinea pig, J. Pharm. Pharmacol. 29, 628. Butterfield, J.L. and K.O. Ellis, 1973, Effects of dantrolene sodium, a skeletal muscle relaxant, on the contractility of cardiac and smooth muscle, Federation Proc. 32, 772. Chipman, M., S. Kaul and M. Lambie, 1974, Efficacy of dantrolene sodium in the treatment of spasticity, Dis. Nerv. Syst. 35,427. Desmedt, J.E. and K. Hainaut, 1977, Inhibition of the intracellular release of calcium by dantrolene in barnacle giant muscle fibers, J. Physiol. 265, 565. Ellis, K.O. and S.H. Bryant, 1972, Excitation--contraction uncoupling in skeletal muscle by dantrolene sodium, Naunyn-Schmiedeb. Arch. Pharmacol. 274, 107. Ellis, K.O. and J.E. Carpenter, 1972, Studies on the mechanism of action of dantrolene sodium a skeletal muscle relaxant, Naunyn-Schmiedeb. Arch. Pharmacol. 275, 83. Ellis, K.O. and J.F. Carperter, 1974, Mechanism of control of skeletal muscle contraction by dantrolene sodium, Arch. Phys. Med. Rehabil. 55, 362. Ellis, K.O., A.W. Castellion, C.J. Honkomp, F.L. Wessels, J.F. Carpenter and R.P. Halliday, 1973, Dantrolene, a direct acting skeletal muscle relaxant, J. Pharm. Sci. 62, 948. Ellis, K.O., J.L. Butterfield, F.L. Wessels and J.F. Carpenter, 1976, A comparison of skeletal, cardiac and smooth muscle actions of dantrolene sodium - - a skeletal muscle relaxant, Arch. Intern. Pharmacodyn. Therap. 244, 1. Ellis, K.O. and F.L. Wessels, 1978, Pharmacological studies of the identified metabolites of dantrolene sodium, Naunyn-Schmiedeb. Arch. Pharmacol. 301,237. Ellis, R.H., P. Simpson, P. Tatham, M. Leighton and J. Williams, 1975, The cardiovascular effect of dantrolene sodium in dogs, Anaesthesia 30, 318. Hainaut, K. and J.E. Desmedt, 1974, Effect of d a n -
342 trolene sodium on calcium movements in single muscle fibers, Nature 252, 5485, p. 728. Harrison, G.G., 1975, Control of the malignant hyperpyrexic syndrome in MHS swine by dantrolene sodium, Brit. J. Anaesth. 47, 62. Herman, R., N. Mayer and S.A. Mecomber, 1972, Clinical pharmaco-physiology of dantrolene sodium, Amer. J. Phys. Med. 51, 6. Hollifield, R.D. and J.D. Conklin, 1973, Determination of dantrolene in biological specimens containing drug-related metabolites, J. Pharm. Sci. 62, 271. Jardon, O.M., D. Kerr, R. Cochram and D.W. Wingard, 1976, Malignant hyperthermia -- a disease of importance to orthopedics (study of dantrolene in porcine malignant hyperthermia), J. Bone Joint Surg. 58, 738. Ladd, H.W., G. Gingras, J. Boucher, N. Kazdan and R. Keenan, 1973, The reductive effect of a new pharmacological agent (dantrolene sodium) on spasticity, Union Med. Can. 102, 1087. Lietman, P.S., R.H.A. Haslam and J.R. Walcher, 1974, Parmacology of dantrolene sodium in children, Arch. Phys. Med. Rehahil. 55, 388.
W.J. MEYLER ET AL. Meyler, W.J., H. Weaseling and S. Agoston, 1976, The effets of dantrolene sodium on cardiac and skeletal muscle in rats, European J. Pharmacol. 39, 127. Monster, A.W., R. Herman, S. Meeks and J. McHenry, 1973, Cooperative study for assessing the effects of a pharmacological agent on spasticity, Amer. J. Phys. Med. 52, 4. Monster, A.W., 1974, Spasticity and the effect of dantrolene sodium, Arch. Phys. Med. Rehab. 55, 373. Morgan, K. and S.H. Bryant, 1977, The mechanism of action of dantrolene sodium, J. Pharmacol. Exptl. Therap. 201, 1. Nott, M.W. and W.C. Bowman, 1974, Actions of dantrolene sodium on contractions of the tibialis anterior and soleus muscles of cats under chloralose anaesthesia, Clin. Exptl. Pharmacol. Physiol. 1,113. Statham, H.E. and C.J. Duncan, 1976, Dantrolene and the neuromuscular junction: Evidence for intracellular calcium stores, European J. Pharmacol. 39, 143. Van Winkle, W.B., 1976, Calcium release from skeletal muscle sarcoplasmatic reticulum: site of action of dantrolene sodium, Science 193, 1130.