Epilepsia, 31(3):313-317, 1990 Raven Press, Ltd., New York 8 International League Against Epilepsy
Comparison of Midazolam and Diazepam by the Intramuscular Route for the Control of Seizures in a Mouse Model of Status Epilepticus Arthur Raines, Theresa R. Henderson, *Ewart A. Swinyard, and Kenneth L. Dretchen Department of Pharmacology, Georgetown University Medical Center, Washington, D.C.; and The Department of Pharmacology and Toxicology, *The University of Utah, Salt Lake City, Utah, U.S.A.
Summary: A model system is described in which sus-
period of -90 min, those treated with DZP 0.2 and 0.4 m a g convulsed for 7.8 and 3.9 min, respectively; mice receiving MDL 0.1 and 0.2 mg/kg convulsed for 1.9 and 1.4 min, respectively. MDL arrested seizures substantially more rapidly than diazepam (p < 0.05). These data
tained clonic seizures are produced by a combination of phenytoin (PHT) and pentylenetetrazol (PTZ) in the mouse, the former agent preventing the terminal tonic spasms produced by the latter. In this system, midazolam (MDL), a water-soluble benzodiazepine, was compared with diazepam (DZP), a sparingly soluble agent which is widely used to treat status epilepticus (SE) in humans. Both agents were administered intramuscularly (i.m.) in approximately equieffective doses in animals exhibiting clonic seizure activity. MDL proved to be about twice as potent as DZP. Whereas control animals convulsed for a
suggest that MDL has sufficiently rapid anticonvulsant action to merit evaluation for control of SE in humans when a rapidly absorbed antiepileptic drug (AED) is needed and intravenous (i.v.) administration is not feasible. Key Words: Neurologic models-convulsionsStatus epilepticus-Midazolam-Diazepam-Anticonvulsants.
AED is needed and i.v. administration is not feasible. Midazolam (MDL) is a recently developed watersoluble benzodiazepine presently used extensively as a preanesthetic medication. heclinical evaluations of the drug indicate that it shares anxiolytic, muscle relaxant, hypnotic, and anticonvulsant actions with other members of this class (Pieri et al., 1981; F’ieri, 1983). Indeed, Pieri et al., (1983) suggested that the drug might be useful as an i.m. agent for treatment of seizures. The drug was reported to suppress interictal spikes in a small series of epileptic patients (Jawad et al., 1986) and therefore may be expected to exert an antiepileptic effect in humans. MDL (as well as other drugs of this class) is a most effective antagonist of pentylenetetrazol (PTZ), and this antagonism was used in develop ment of a model for SE that could be tested in a treatment mode; i.e., we developed a simple method which reliably yielded convulsive seizures of sufficient duration that the efficacy of an i.m. intervention could be assessed. In this setting, we
Although benzodiazepines as a group have not yielded primary antiepileptic drugs (AEDs), diazepam (DZP) is regarded by many researchers to be a drug of choice for emergency treatment of status epilepticus (SE). Intravenous (i.v.) administration is by far the preferred route in this setting, as intramuscularly (i.m.) injected DZP is only slowly absorbed and produces relatively low serum levels (Hillestad et al., 1974). The reason for this lies in the very limited aqueous solubility of DZP, leading to formation of a depot of sparingly soluble material. This slowly mobilized depot makes achievement of adequate brain concentrations difficult in the face of redistribution of DZP. This is unfortunate because circumstances often exist when a rapidly absorbed Received December 1988; revision accepted March 1989. A preliminary report of this work was presented at the December, I987 meeting of the American Epilepsy Society, Baltimore, MD, U.S.A., and published in part in Epilepsia 1987; 28:632. Address correspondence and reprint requests to Dr.A. Raines at Department of Pharmacology, Georgetown University Medical Center, Washington, D.C. 20007, U.S.A.
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314
compared the speed and efficacy of the anticonvulsant effect of MDL with that of DZP.
METHODS Male Swiss-Webster mice weighing 2&25 g were used in these studies. All animals were housed in group cages containing eight mice each and allowed free access to food and water. The animals were moved to the laboratory 24 h before the experiment and were permitted to acclimatize to their surroundings. For the standard convulsive stimulus, the following procedure was used: a 50-mgkg dose of phenytoin (PHT) sodium (Parke-Davis, Detroit, MI, U.S.A.) dissolved in alkalinized saline solution was administered intrapentoneally (i.p.) in a volume of 0.1 mVlO g body weight; 2 h later, PTZ (Sigma Chemical, St. Louis, MO, U.S.A.) dissolved in saline solution, was administered subcutaneously (s.c.) in a dose of 100 mgkg. The injection was made in the loose skin behind the neck in a volume of 0.01 mVlO g body weight. The time needed for development of unequivocal sustained clonic seizure activity involving the limbs (isolated myoclonic jerks or other preconvulsive chewing behavior were not counted) was carefully timed, and treatment was initiated within 18 s. The latter consisted of an intramuscular injection of either saline solution, DZP or MDL. These preparations were injected into a hindlimb thigh muscle in a volume of 0.01 m1/10 g body weight. The benzodiazepine drugs (Roche Laboratories, Nutley NJ, U.S.A.) were obtained from the hospital pharmacy in the form of their respective commercially available pare n t e d solutions and diluted with either saline solution (MDL) or a solution containing 40% propylene glycol, 10% ethanol, and 50% saline (DZP). Both benzodiazepine drugs were administered in doses based on their free base content (as labeled by the manufacturer). Several doses of MDL and DZP were used in these studies. The time necessary for the seizures to be brought under control was carefully timed for each treatment, and the behavior of the animals with regard to locomotion and lethargy was assessed. Development of “breakthrough seizures” after control had apparently been achieved was also noted. Mice in which breakthrough seizures occurred were regarded as not having achieved lasting (defined as at least 1 h) control. The incidence of fatalities resulting from seizures was determined. The data were subjected to an analysis of variance, and differences with a p value of 50 mgkg produced motor disturbances consisting of tremor, clonic motor movements, and gait abnormalities that interfered with precise identification of onset of seizure activity after PTZ injection. At a dose of 50 mgkg, the mice exhibited normal motor activity and no instances of tonic hindlimb extension were observed after PTZ administration. Thus, the model of SE was satisfactory for the purposes of the present investigation as the animals were asymptomatic until after receiving the PTZ. Subsequent to PTZ administration, unequivocal clonic seizure activity began in 4.51 rt 0.318 min (SEM). In animals receiving treatment with i.m. saline solution, the duration of the seizures ranged from 34 to >150 min. The mean seizure duration in the 13 mice that survived the seizures was 90 5 9.42 min (SEM); seven mice died; these latter animals convulsed for a mean duration of 21.4 min before succumbing to the clonic seizure episode. None of the animals experienced a tonic hindlimb extension, which is a frequent terminal event of seizures produced by large doses of PTZ. In preliminary dose-range studies, in which it was necessary to identify approximately equieffective doses and appropriate ratios for the two benzodiazepines, we evaluated six doses of DZP (0.05-5.0 mg/kg) and five doses of MDL (0.0054.20 mg/kg) in groups of 10-14 mice each. The endpoints sought were termination of clonic seizures and the capacity to maintain a seizure-free state (i.e., without breakthrough seizures) for a period of at least 1 h (lasting control). The results of these studies are shown in Table 1. Doses of DZP of 0.5-5.0 mg/kg, although TABLE 1. Eflcacy of anticonvulsant treatments Dose (mg/kg)
Seizure control
Lasting control“
(%)
(%)
Diazepam 0.05 0.10
0.20 0.40 0.50 5.0
20 0
12 100 100 100
Midazolam
0
38 100 100
100
0.005
0
-
0.025
40
20
0.05 0.10
9o
60 9o
0.20 a
100 100
Defined as no seizures for at least 1 h.
83
MDL A N D DZP IN SE IN MICE quite effective, were obviously sedating, with animals at the higher dose exhibiting little or no locomotor behavior. Doses of DZP of 0.2 and 0.4 mg/kg proved to be both effective and not overtly sedating. Similarly, doses of MDL which were found to be approximately as equieffective as the DZP doses, were 0.1 and 0.2 mg/kg. Doses
Comparison of Midazolam and Diazepam by the Intramuscular Route for the Control of Seizures in a Mouse Model of Status Epilepticus Arthur Raines, Theresa R. Henderson, *Ewart A. Swinyard, and Kenneth L. Dretchen Department of Pharmacology, Georgetown University Medical Center, Washington, D.C.; and The Department of Pharmacology and Toxicology, *The University of Utah, Salt Lake City, Utah, U.S.A.
Summary: A model system is described in which sus-
period of -90 min, those treated with DZP 0.2 and 0.4 m a g convulsed for 7.8 and 3.9 min, respectively; mice receiving MDL 0.1 and 0.2 mg/kg convulsed for 1.9 and 1.4 min, respectively. MDL arrested seizures substantially more rapidly than diazepam (p < 0.05). These data
tained clonic seizures are produced by a combination of phenytoin (PHT) and pentylenetetrazol (PTZ) in the mouse, the former agent preventing the terminal tonic spasms produced by the latter. In this system, midazolam (MDL), a water-soluble benzodiazepine, was compared with diazepam (DZP), a sparingly soluble agent which is widely used to treat status epilepticus (SE) in humans. Both agents were administered intramuscularly (i.m.) in approximately equieffective doses in animals exhibiting clonic seizure activity. MDL proved to be about twice as potent as DZP. Whereas control animals convulsed for a
suggest that MDL has sufficiently rapid anticonvulsant action to merit evaluation for control of SE in humans when a rapidly absorbed antiepileptic drug (AED) is needed and intravenous (i.v.) administration is not feasible. Key Words: Neurologic models-convulsionsStatus epilepticus-Midazolam-Diazepam-Anticonvulsants.
AED is needed and i.v. administration is not feasible. Midazolam (MDL) is a recently developed watersoluble benzodiazepine presently used extensively as a preanesthetic medication. heclinical evaluations of the drug indicate that it shares anxiolytic, muscle relaxant, hypnotic, and anticonvulsant actions with other members of this class (Pieri et al., 1981; F’ieri, 1983). Indeed, Pieri et al., (1983) suggested that the drug might be useful as an i.m. agent for treatment of seizures. The drug was reported to suppress interictal spikes in a small series of epileptic patients (Jawad et al., 1986) and therefore may be expected to exert an antiepileptic effect in humans. MDL (as well as other drugs of this class) is a most effective antagonist of pentylenetetrazol (PTZ), and this antagonism was used in develop ment of a model for SE that could be tested in a treatment mode; i.e., we developed a simple method which reliably yielded convulsive seizures of sufficient duration that the efficacy of an i.m. intervention could be assessed. In this setting, we
Although benzodiazepines as a group have not yielded primary antiepileptic drugs (AEDs), diazepam (DZP) is regarded by many researchers to be a drug of choice for emergency treatment of status epilepticus (SE). Intravenous (i.v.) administration is by far the preferred route in this setting, as intramuscularly (i.m.) injected DZP is only slowly absorbed and produces relatively low serum levels (Hillestad et al., 1974). The reason for this lies in the very limited aqueous solubility of DZP, leading to formation of a depot of sparingly soluble material. This slowly mobilized depot makes achievement of adequate brain concentrations difficult in the face of redistribution of DZP. This is unfortunate because circumstances often exist when a rapidly absorbed Received December 1988; revision accepted March 1989. A preliminary report of this work was presented at the December, I987 meeting of the American Epilepsy Society, Baltimore, MD, U.S.A., and published in part in Epilepsia 1987; 28:632. Address correspondence and reprint requests to Dr.A. Raines at Department of Pharmacology, Georgetown University Medical Center, Washington, D.C. 20007, U.S.A.
313
A . RAINES ET AL.
314
compared the speed and efficacy of the anticonvulsant effect of MDL with that of DZP.
METHODS Male Swiss-Webster mice weighing 2&25 g were used in these studies. All animals were housed in group cages containing eight mice each and allowed free access to food and water. The animals were moved to the laboratory 24 h before the experiment and were permitted to acclimatize to their surroundings. For the standard convulsive stimulus, the following procedure was used: a 50-mgkg dose of phenytoin (PHT) sodium (Parke-Davis, Detroit, MI, U.S.A.) dissolved in alkalinized saline solution was administered intrapentoneally (i.p.) in a volume of 0.1 mVlO g body weight; 2 h later, PTZ (Sigma Chemical, St. Louis, MO, U.S.A.) dissolved in saline solution, was administered subcutaneously (s.c.) in a dose of 100 mgkg. The injection was made in the loose skin behind the neck in a volume of 0.01 mVlO g body weight. The time needed for development of unequivocal sustained clonic seizure activity involving the limbs (isolated myoclonic jerks or other preconvulsive chewing behavior were not counted) was carefully timed, and treatment was initiated within 18 s. The latter consisted of an intramuscular injection of either saline solution, DZP or MDL. These preparations were injected into a hindlimb thigh muscle in a volume of 0.01 m1/10 g body weight. The benzodiazepine drugs (Roche Laboratories, Nutley NJ, U.S.A.) were obtained from the hospital pharmacy in the form of their respective commercially available pare n t e d solutions and diluted with either saline solution (MDL) or a solution containing 40% propylene glycol, 10% ethanol, and 50% saline (DZP). Both benzodiazepine drugs were administered in doses based on their free base content (as labeled by the manufacturer). Several doses of MDL and DZP were used in these studies. The time necessary for the seizures to be brought under control was carefully timed for each treatment, and the behavior of the animals with regard to locomotion and lethargy was assessed. Development of “breakthrough seizures” after control had apparently been achieved was also noted. Mice in which breakthrough seizures occurred were regarded as not having achieved lasting (defined as at least 1 h) control. The incidence of fatalities resulting from seizures was determined. The data were subjected to an analysis of variance, and differences with a p value of 50 mgkg produced motor disturbances consisting of tremor, clonic motor movements, and gait abnormalities that interfered with precise identification of onset of seizure activity after PTZ injection. At a dose of 50 mgkg, the mice exhibited normal motor activity and no instances of tonic hindlimb extension were observed after PTZ administration. Thus, the model of SE was satisfactory for the purposes of the present investigation as the animals were asymptomatic until after receiving the PTZ. Subsequent to PTZ administration, unequivocal clonic seizure activity began in 4.51 rt 0.318 min (SEM). In animals receiving treatment with i.m. saline solution, the duration of the seizures ranged from 34 to >150 min. The mean seizure duration in the 13 mice that survived the seizures was 90 5 9.42 min (SEM); seven mice died; these latter animals convulsed for a mean duration of 21.4 min before succumbing to the clonic seizure episode. None of the animals experienced a tonic hindlimb extension, which is a frequent terminal event of seizures produced by large doses of PTZ. In preliminary dose-range studies, in which it was necessary to identify approximately equieffective doses and appropriate ratios for the two benzodiazepines, we evaluated six doses of DZP (0.05-5.0 mg/kg) and five doses of MDL (0.0054.20 mg/kg) in groups of 10-14 mice each. The endpoints sought were termination of clonic seizures and the capacity to maintain a seizure-free state (i.e., without breakthrough seizures) for a period of at least 1 h (lasting control). The results of these studies are shown in Table 1. Doses of DZP of 0.5-5.0 mg/kg, although TABLE 1. Eflcacy of anticonvulsant treatments Dose (mg/kg)
Seizure control
Lasting control“
(%)
(%)
Diazepam 0.05 0.10
0.20 0.40 0.50 5.0
20 0
12 100 100 100
Midazolam
0
38 100 100
100
0.005
0
-
0.025
40
20
0.05 0.10
9o
60 9o
0.20 a
100 100
Defined as no seizures for at least 1 h.
83
MDL A N D DZP IN SE IN MICE quite effective, were obviously sedating, with animals at the higher dose exhibiting little or no locomotor behavior. Doses of DZP of 0.2 and 0.4 mg/kg proved to be both effective and not overtly sedating. Similarly, doses of MDL which were found to be approximately as equieffective as the DZP doses, were 0.1 and 0.2 mg/kg. Doses
