Psychopharmacologia (Berl.) 41, 305-308 (1975) 9 by Springer-Verlag 1975
Short Reports
The Effect of Ouabain and Digitoxin on Hexobarbitone Sleeping Time in the Mouse NEIL S. DOGGETT Department of Applied Pharmacology, Welsh School of Pharmacy, UWIST, Cardiff, Great Britain Received November 12, 1974; Final Version December 9, 1974
Abstract. The effect of ouabain and digitoxin upon the duration of loss of righting reflex produced by hexobarbitone in mice pretreated with the liver microsomal enzyme inhibitor SKF 525A was studied. Both ouabain and, to a greater extent digitoxin, produced a dose dependent potentiation
of the activity of hexobarbitone. These results confirm and extend previous observations and may be explained by a direct interaction at the level of the CNS and by a modified distribution of the barbiturate. Furthermore, they re-emphasize the possibility of such an interaction in the clinic.
Key words: Cardiac Glycosides - Hexobarbitone - Sleeping Time.
Introduction It has been well documented over the years that the symptoms of cardiac glycoside administration in man include depressant effects upon the CNS. First noted by Withering (1785), they have been repeatedly seen by many workers (see Batterman and Gutner, 1948). These neurological symptoms may be the earliest, sometimes the most severe and perhaps the only component of digitalis intoxication, but it is not known with certainty whether they are mediated directly or if they are behavioural manifestations of peripheral activity. Until comparatively recently, the effects of cardiac glycosides on the CNS of laboratory rodents were thought to be predominantly excitatory, ultimately giving rise to convulsions and death (Greef and Kasperat, 1961a, b). However, in 1970 Doggett, Spencer and Turner demonstrated a biphasic response to ouabain and, in addition to the more usual excitatory component, showed that small doses (0.1-0.4 gg) produced a behavioural depression when injected into the cerebral ventricles of conscious mice. Furthermore, these experiments, together with others in rats (Doggett and Spencer, 1973) and chickens (Doggett
and Spencer, 1972) have revealed that these depressant effects are mediated through a direct action on the CNS. Small doses of centrally administered ouabain can also markedly potentiate the hypnotic activity of pentobarbitone (Doggett, 1973) and preliminary observations (Ferrini and Scuka, 1967) have indicated that a similar potentiation of pentobarbitone and quinalbarbitone can occur after the peripheral (oral) administration of several different glycosides. However, in view of the inhibitory effect of cardiac glycosides upon microsomal enzyme systems, it was necessary to eliminate the possibility that a decrease in barbiturate metabolism was responsible for their observed potentiation. Consequently this study investigates the ability of two glycosides with different pharmacokinetic properties to potentiate hexobarbitone in mice pretreated with SKF 525A. Methods Male albino mice of an ICI strain, weighing 20 + 2 g were housed under constant environmental conditions. They were allowed access to drinking water and 41B cube diet until 2 hrs before commencing the experiment, when they were
306
Psychopharmacologia (Berl.), Vol. 41, Fasc. 3 (1975)
pretreated with SKF 525A (50 mg/kg s.c.), randomly divided into groups of ten and transferred to the laboratory which was maintained at a constant temperature of 25 + 1 ~C. Food and water were withdrawn immediately before the experiment. The hypnotic activity induced by hexobarbitone was recorded as the duration of a complete loss of righting reflex, the results being expressed as the group mean sleeping time + S.E.M. The statistical significance of any observed differences were calculated using Student's t test. In several animals the electrocardiogram (ECG) was recorded after intraperitoneal cardiac glycoside administration, the standard lead II being monitored continuously on a Devices MX2 recorder using subcutaneous electrodes. Hexobarbitone, as the sodium salt, and SKF 525A were dissolved in 0.9% sterile apyrogenic saline. Ouabain and digitoxin were dissolved in a mixture of 5 % ethanol, 5 % glycerin and 0.9 % sterile apyrogenic saline. The dose vol: ume in all cases was 10 ml/kg.
3500-
3000-
Ld
2600 -
bO
(J LLI UO
2000 -
LLI
_:Z: 1500 z [3_ LLI LLI
1000-
500-
Results Effect of Ouabain and Digitoxin on the Dose-Response Curve of Hexobarbitone. Three groups of ten SKF 525A pretreated mice received either ouabain (0.5 mg/kg i.p.), digitoxin (0.5 mg/kg i.p.), or vehicle 30 min before various doses of hexobarbitone (58-80 mg/kg i.p.). Over this dose range it was found that both ouabain and digitoxin markedly potentiate the duration of hexobarbitone sleeping time. This resulted in parallel shifts to the left of the linear portion of the log dose-response curve, and an increase in potency by a factor of 1.7 in the presence of ouabain and 2.8 with digitoxin (Fig. 1).
Determination of Threshold Dose and Dose-Response Relationship for Cardiac Glycosides. In order to detect the minimum doses of ouabain and digitoxin necessary to produce a significant potentiation of hexobarbitone sleeping time and in order to determine whether a dose-response relationship existed, a log dose response curve was constructed for both drugs by measuring the sleeping times produced by a single suprathreshold, sub-maximal dose of hexobarbitone when administered after various doses of each agent. Two groups of ten SKF 525A pretreated mice received ouabain or digitoxin (0.1-2.0 mg/kg i.p.) 30 min before hexobarbitone (65 mg/kg i.p.). One additional pretreated group received vehicle alone 30 min before the same dose of hexobarbitone and served as control. The sleeping time in this control group was 578 • 12 sec. It was found that a minimum dose of 0.25 mg/kg of both ouabain and digitoxin was required in order to produce a significant (P < 0.05) potentiation of hexobarbitone sleeping time. Furthermore, this effect
II0
I
20
iO
810
I
HEXOBARBiTONE (mg/kg)
Fig. 1. Potentiation of hexobarbitone dose-response curve by ouabain and digitoxin. Animals received saline, 10 ml/kg (X x ), ouabain, 0.5 mg/kg (O 0), or digitoxin, 0.5 mg/kg (9 9 30 min before various doses of hexobarbitone. All animals were pretreated with SKF 525A (50 mg/kg s.c.) 2 hrs before commencing the experiment
was dose-related, with digitoxin being more potent than ouabain (Fig. 2). It was also noted that the toxicity of hexobarbitone was markedly increased when doses of cardiac glycoside above 2.0 mg/kg were administered.
Effect of Ouabain and Digitoxin on the Mouse Heart. In view of the relatively large (cf. man) doses employed, it was necessary to eliminate the possibility that a disturbance in cardiac function resulting in cerebral hypoxia was contributing to the observed potentiation. Consequently, the E C G was recorded in several animals following various doses of each cardiac glycoside. Groups of five SKF 525A pretreated mice received ouabain or digitoxin (0.25, 0.5, 1.0 and 2.0 mg/kg i.p.) and the E C G was monitored continuously. Further groups received the glycosides at the above doses followed 30 min later by hexobarbitone (80 mg/kg i.p.). No evidence of any cardiotoxic effects or embarrassment of cardiac function was obtained from any of the electrical records in the above cases.
N. S. Doggett: Hexobarbitone Potentiation by Cardiac Glycosides
2400
LLi Ul 4~
2000
LLI U3 LLI ~E
1600' Z LU LLI
120C
800
011
025
0!5
110
20
DOSE ( m g / k g )
Fig. 2. Effect of 30 rain pretreatment with various doses of ouabain ( I Q) and digitoxin ( O O) on the sleeping time induced by a singIe dose (65 mg/kg) of hexobarbitone. All animals received SKF 525A (50 mg/kg s.c.) 2 hrs before commencing the experiment
Discussion It has already been shown in mice that at least one component of the central depressant effects of the cardiac glycosides is brought about by a direct effect within the CNS (Doggett et al., 1970), and that following intracerebroventricular injection, these drugs potentiate the hypnotic effect of pentobarbitone (Doggett, 1973). Since digitalis glycosides are frequently given together with sedative drugs, including the barbiturates, it was important to determine if sufficient glycoside could reach the CNS after peripheral administration to potentiate the barbiturate effect. Although Ferrine and Scuka (1967) have already shown that such a potentiation does indeed occur following oral administration of desacetyl lanatoside C, ouabain and adonis glycosides, they were unable to eliminate an effect upon liver microsomal enzymes resuiting in a decrease in the rate of barbiturate metabolism as being responsible for the observed potentiation. The results presented here, however, using hexobarbitone in mice pretreated with SKF 525A
307
demonstrate conclusively that a glycoside induced barbiturate potentiation can occur independently of any metabolic interference, the effect of both glycosides studied being dose dependent. The increased potency of digitoxin compared with that of ouabain may well result from the ease with which each compound can cross the blood-brain barrier and consequently gain access to central sites, as it reflects the different lipid solubilities of the two compounds. But, since termination of the effect of hexobarbitone in SKF 525A pretreated animals is determined solely by redistribution, principally within fat depots within the body, it may be argued that some component of the effect reported here arises from a displacement of barbiturate from these fat stores. Similarly, it may be said that the glycosides displace the barbiturate from plasma proteins to which it is reversibly bound (Goldbaum and Smith, 1954). Whereas these arguments may be considered of some relevance in the case of digitoxin, and may account for its increased potency, they would not be expected to be important in explaining the activity of ouabain which is neither appreciably lipid soluble nor bound by plasma proteins (Scholtan et al., 1966). Consequently, although an effect upon barbiturate distribution may in some cases take place, these results give strong support to the suggestion that a direct interaction within the CNS does occur following peripheral administration of cardiac glycosides. It is appreciated that the doses used in these experiments are larger than those in routine clinical use, representing for ouabain a twenty fold and for digitoxin a ten fold increase in the maximum recommended digitalising doses. Nevertheless, the ability of these compounds to produce significant central effects after peripheral injection of non-toxic doses in laboratory animals emphasizes the need for the clinician to be alert to the possibility of enhanced effects of concurrently administered sedatives in digitalised patients.
References Batterman, R.C., Gutner, L.B.: Hitherto undescribed neurological manifestations of digitalis toxicity. Amer. Heart J. 36, 582-586 (1948) Doggett, N. S.: Interaction of ouabain with pentobarbitone in the mouse. Arch. int. Pharmacodyn. 204, 56-61 (~973) Doggett, N. S., Spencer, P. S. J.: Ouabain and the onset of the blood-brain barrier in neonate chicks. Nature (Lond.) 237, 513-514 (1972) Doggett, N. S., Spencer, P. S. J.: Pharmacological properties of centralIy administered agents which interfere with neurotransmitter function: A comparison with the central depressant effects of ouahain. Brit. J. Pharmacol. 47, 26-38 (1973)
308 Doggett, N. S., Spencer, P. S.J., Turner, T. A.R.: The pharmacological effects of ouabain administered intracerebrally to conscious mice. Brit. J. Pharmacol. 40, 138P (1970) Ferrini, R., Scuka, M.: Increased barbiturate sleeping time by simultaneous administration of cardiac glycosides to mice. J. Pharmac. Pharmacol. 19, 780 (1967) Goldbaum, L. R., Smith, P. K.: The interaction of barbiturates with serum albumin and its possible relation to their disposition and pharmacological actions. J. Pharmacol. exp. Ther. 111, 197-209 (1954) Greef, K., Kasperat, H.: Konvulsive und paralytische Wirkungen von Digitalis-Glykosiden und Geninen bei in-
Dr. N. S. Doggett, Department of Applied Pharmacology Welsh School of Pharmacy, UWIST, Cardiff, Great Britain
Psychopharmacologia (Berl.), Vol. 41, Fasc. 3 (1975) tracerebraler und intraven6ser Injektion an M~iusen. Arzneimittel-Forsch. 11, 908-909 (1961a) Greef, K., Kasperat, H.: Vergleich der neurotoxischen Wirkung von Digitalisglykosiden und Geninen bei intracerebraler und intraventser Injektion an M~iusen, Ratten und Meerschweinchen. Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak. 242, 76-89 (1961b) Scholtan, W., Schlossman, K., Rosenkranz, H.: Bestimmung der EiweiBbindung von Digitalis-Pr~iparaten mittels der Ultracentrifuge. Arzneimittel-Forsch. 16, 109-118 (1966) Withering, W.: An account of the foxglove and some of its medicinal uses: with practical remarks on dropsy and other diseases. Birmingham: M. Swinney 1785
Short Reports
The Effect of Ouabain and Digitoxin on Hexobarbitone Sleeping Time in the Mouse NEIL S. DOGGETT Department of Applied Pharmacology, Welsh School of Pharmacy, UWIST, Cardiff, Great Britain Received November 12, 1974; Final Version December 9, 1974
Abstract. The effect of ouabain and digitoxin upon the duration of loss of righting reflex produced by hexobarbitone in mice pretreated with the liver microsomal enzyme inhibitor SKF 525A was studied. Both ouabain and, to a greater extent digitoxin, produced a dose dependent potentiation
of the activity of hexobarbitone. These results confirm and extend previous observations and may be explained by a direct interaction at the level of the CNS and by a modified distribution of the barbiturate. Furthermore, they re-emphasize the possibility of such an interaction in the clinic.
Key words: Cardiac Glycosides - Hexobarbitone - Sleeping Time.
Introduction It has been well documented over the years that the symptoms of cardiac glycoside administration in man include depressant effects upon the CNS. First noted by Withering (1785), they have been repeatedly seen by many workers (see Batterman and Gutner, 1948). These neurological symptoms may be the earliest, sometimes the most severe and perhaps the only component of digitalis intoxication, but it is not known with certainty whether they are mediated directly or if they are behavioural manifestations of peripheral activity. Until comparatively recently, the effects of cardiac glycosides on the CNS of laboratory rodents were thought to be predominantly excitatory, ultimately giving rise to convulsions and death (Greef and Kasperat, 1961a, b). However, in 1970 Doggett, Spencer and Turner demonstrated a biphasic response to ouabain and, in addition to the more usual excitatory component, showed that small doses (0.1-0.4 gg) produced a behavioural depression when injected into the cerebral ventricles of conscious mice. Furthermore, these experiments, together with others in rats (Doggett and Spencer, 1973) and chickens (Doggett
and Spencer, 1972) have revealed that these depressant effects are mediated through a direct action on the CNS. Small doses of centrally administered ouabain can also markedly potentiate the hypnotic activity of pentobarbitone (Doggett, 1973) and preliminary observations (Ferrini and Scuka, 1967) have indicated that a similar potentiation of pentobarbitone and quinalbarbitone can occur after the peripheral (oral) administration of several different glycosides. However, in view of the inhibitory effect of cardiac glycosides upon microsomal enzyme systems, it was necessary to eliminate the possibility that a decrease in barbiturate metabolism was responsible for their observed potentiation. Consequently this study investigates the ability of two glycosides with different pharmacokinetic properties to potentiate hexobarbitone in mice pretreated with SKF 525A. Methods Male albino mice of an ICI strain, weighing 20 + 2 g were housed under constant environmental conditions. They were allowed access to drinking water and 41B cube diet until 2 hrs before commencing the experiment, when they were
306
Psychopharmacologia (Berl.), Vol. 41, Fasc. 3 (1975)
pretreated with SKF 525A (50 mg/kg s.c.), randomly divided into groups of ten and transferred to the laboratory which was maintained at a constant temperature of 25 + 1 ~C. Food and water were withdrawn immediately before the experiment. The hypnotic activity induced by hexobarbitone was recorded as the duration of a complete loss of righting reflex, the results being expressed as the group mean sleeping time + S.E.M. The statistical significance of any observed differences were calculated using Student's t test. In several animals the electrocardiogram (ECG) was recorded after intraperitoneal cardiac glycoside administration, the standard lead II being monitored continuously on a Devices MX2 recorder using subcutaneous electrodes. Hexobarbitone, as the sodium salt, and SKF 525A were dissolved in 0.9% sterile apyrogenic saline. Ouabain and digitoxin were dissolved in a mixture of 5 % ethanol, 5 % glycerin and 0.9 % sterile apyrogenic saline. The dose vol: ume in all cases was 10 ml/kg.
3500-
3000-
Ld
2600 -
bO
(J LLI UO
2000 -
LLI
_:Z: 1500 z [3_ LLI LLI
1000-
500-
Results Effect of Ouabain and Digitoxin on the Dose-Response Curve of Hexobarbitone. Three groups of ten SKF 525A pretreated mice received either ouabain (0.5 mg/kg i.p.), digitoxin (0.5 mg/kg i.p.), or vehicle 30 min before various doses of hexobarbitone (58-80 mg/kg i.p.). Over this dose range it was found that both ouabain and digitoxin markedly potentiate the duration of hexobarbitone sleeping time. This resulted in parallel shifts to the left of the linear portion of the log dose-response curve, and an increase in potency by a factor of 1.7 in the presence of ouabain and 2.8 with digitoxin (Fig. 1).
Determination of Threshold Dose and Dose-Response Relationship for Cardiac Glycosides. In order to detect the minimum doses of ouabain and digitoxin necessary to produce a significant potentiation of hexobarbitone sleeping time and in order to determine whether a dose-response relationship existed, a log dose response curve was constructed for both drugs by measuring the sleeping times produced by a single suprathreshold, sub-maximal dose of hexobarbitone when administered after various doses of each agent. Two groups of ten SKF 525A pretreated mice received ouabain or digitoxin (0.1-2.0 mg/kg i.p.) 30 min before hexobarbitone (65 mg/kg i.p.). One additional pretreated group received vehicle alone 30 min before the same dose of hexobarbitone and served as control. The sleeping time in this control group was 578 • 12 sec. It was found that a minimum dose of 0.25 mg/kg of both ouabain and digitoxin was required in order to produce a significant (P < 0.05) potentiation of hexobarbitone sleeping time. Furthermore, this effect
II0
I
20
iO
810
I
HEXOBARBiTONE (mg/kg)
Fig. 1. Potentiation of hexobarbitone dose-response curve by ouabain and digitoxin. Animals received saline, 10 ml/kg (X x ), ouabain, 0.5 mg/kg (O 0), or digitoxin, 0.5 mg/kg (9 9 30 min before various doses of hexobarbitone. All animals were pretreated with SKF 525A (50 mg/kg s.c.) 2 hrs before commencing the experiment
was dose-related, with digitoxin being more potent than ouabain (Fig. 2). It was also noted that the toxicity of hexobarbitone was markedly increased when doses of cardiac glycoside above 2.0 mg/kg were administered.
Effect of Ouabain and Digitoxin on the Mouse Heart. In view of the relatively large (cf. man) doses employed, it was necessary to eliminate the possibility that a disturbance in cardiac function resulting in cerebral hypoxia was contributing to the observed potentiation. Consequently, the E C G was recorded in several animals following various doses of each cardiac glycoside. Groups of five SKF 525A pretreated mice received ouabain or digitoxin (0.25, 0.5, 1.0 and 2.0 mg/kg i.p.) and the E C G was monitored continuously. Further groups received the glycosides at the above doses followed 30 min later by hexobarbitone (80 mg/kg i.p.). No evidence of any cardiotoxic effects or embarrassment of cardiac function was obtained from any of the electrical records in the above cases.
N. S. Doggett: Hexobarbitone Potentiation by Cardiac Glycosides
2400
LLi Ul 4~
2000
LLI U3 LLI ~E
1600' Z LU LLI
120C
800
011
025
0!5
110
20
DOSE ( m g / k g )
Fig. 2. Effect of 30 rain pretreatment with various doses of ouabain ( I Q) and digitoxin ( O O) on the sleeping time induced by a singIe dose (65 mg/kg) of hexobarbitone. All animals received SKF 525A (50 mg/kg s.c.) 2 hrs before commencing the experiment
Discussion It has already been shown in mice that at least one component of the central depressant effects of the cardiac glycosides is brought about by a direct effect within the CNS (Doggett et al., 1970), and that following intracerebroventricular injection, these drugs potentiate the hypnotic effect of pentobarbitone (Doggett, 1973). Since digitalis glycosides are frequently given together with sedative drugs, including the barbiturates, it was important to determine if sufficient glycoside could reach the CNS after peripheral administration to potentiate the barbiturate effect. Although Ferrine and Scuka (1967) have already shown that such a potentiation does indeed occur following oral administration of desacetyl lanatoside C, ouabain and adonis glycosides, they were unable to eliminate an effect upon liver microsomal enzymes resuiting in a decrease in the rate of barbiturate metabolism as being responsible for the observed potentiation. The results presented here, however, using hexobarbitone in mice pretreated with SKF 525A
307
demonstrate conclusively that a glycoside induced barbiturate potentiation can occur independently of any metabolic interference, the effect of both glycosides studied being dose dependent. The increased potency of digitoxin compared with that of ouabain may well result from the ease with which each compound can cross the blood-brain barrier and consequently gain access to central sites, as it reflects the different lipid solubilities of the two compounds. But, since termination of the effect of hexobarbitone in SKF 525A pretreated animals is determined solely by redistribution, principally within fat depots within the body, it may be argued that some component of the effect reported here arises from a displacement of barbiturate from these fat stores. Similarly, it may be said that the glycosides displace the barbiturate from plasma proteins to which it is reversibly bound (Goldbaum and Smith, 1954). Whereas these arguments may be considered of some relevance in the case of digitoxin, and may account for its increased potency, they would not be expected to be important in explaining the activity of ouabain which is neither appreciably lipid soluble nor bound by plasma proteins (Scholtan et al., 1966). Consequently, although an effect upon barbiturate distribution may in some cases take place, these results give strong support to the suggestion that a direct interaction within the CNS does occur following peripheral administration of cardiac glycosides. It is appreciated that the doses used in these experiments are larger than those in routine clinical use, representing for ouabain a twenty fold and for digitoxin a ten fold increase in the maximum recommended digitalising doses. Nevertheless, the ability of these compounds to produce significant central effects after peripheral injection of non-toxic doses in laboratory animals emphasizes the need for the clinician to be alert to the possibility of enhanced effects of concurrently administered sedatives in digitalised patients.
References Batterman, R.C., Gutner, L.B.: Hitherto undescribed neurological manifestations of digitalis toxicity. Amer. Heart J. 36, 582-586 (1948) Doggett, N. S.: Interaction of ouabain with pentobarbitone in the mouse. Arch. int. Pharmacodyn. 204, 56-61 (~973) Doggett, N. S., Spencer, P. S. J.: Ouabain and the onset of the blood-brain barrier in neonate chicks. Nature (Lond.) 237, 513-514 (1972) Doggett, N. S., Spencer, P. S. J.: Pharmacological properties of centralIy administered agents which interfere with neurotransmitter function: A comparison with the central depressant effects of ouahain. Brit. J. Pharmacol. 47, 26-38 (1973)
308 Doggett, N. S., Spencer, P. S.J., Turner, T. A.R.: The pharmacological effects of ouabain administered intracerebrally to conscious mice. Brit. J. Pharmacol. 40, 138P (1970) Ferrini, R., Scuka, M.: Increased barbiturate sleeping time by simultaneous administration of cardiac glycosides to mice. J. Pharmac. Pharmacol. 19, 780 (1967) Goldbaum, L. R., Smith, P. K.: The interaction of barbiturates with serum albumin and its possible relation to their disposition and pharmacological actions. J. Pharmacol. exp. Ther. 111, 197-209 (1954) Greef, K., Kasperat, H.: Konvulsive und paralytische Wirkungen von Digitalis-Glykosiden und Geninen bei in-
Dr. N. S. Doggett, Department of Applied Pharmacology Welsh School of Pharmacy, UWIST, Cardiff, Great Britain
Psychopharmacologia (Berl.), Vol. 41, Fasc. 3 (1975) tracerebraler und intraven6ser Injektion an M~iusen. Arzneimittel-Forsch. 11, 908-909 (1961a) Greef, K., Kasperat, H.: Vergleich der neurotoxischen Wirkung von Digitalisglykosiden und Geninen bei intracerebraler und intraventser Injektion an M~iusen, Ratten und Meerschweinchen. Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak. 242, 76-89 (1961b) Scholtan, W., Schlossman, K., Rosenkranz, H.: Bestimmung der EiweiBbindung von Digitalis-Pr~iparaten mittels der Ultracentrifuge. Arzneimittel-Forsch. 16, 109-118 (1966) Withering, W.: An account of the foxglove and some of its medicinal uses: with practical remarks on dropsy and other diseases. Birmingham: M. Swinney 1785
