Acta pharmacol. et toxicol. 1975, 36,3 12-320.
From the Department of Pharmacology, Faculty of Pharmacy, University of Uppsala, Box 573, S-751 23 Uppsala, Sweden
Anticonvulsant Activity in Mice of Diazepam in an Emulsion Formulation for Intravenous Administration BY Roland Jeppsson and Stellan Ljungberg (Received October 23, 1974; Accepted November 4, 1974)
Abstract: The anticonvulsant activity of diazepam has been investigated in mice using an electrical low voltage square wave technique. Diazepam was administered intravenously using two injection formulations, Valium@inj. (Roche) and diazepam emulsion. In the latter formulation the lipophilic drug was dissolved in soya bean oil which was then emulsified to an o/w emulsion. A great difference was found in acute intravenous toxicity (LD50) between the two diazepam formulations. The higher toxicity of Valium" inj. could probably be due to the solvents used in this formulation. The anticonvulsant activity was about equal for the two preparations. Thus, no difference was found in duration of anticonvulsant activity at the two dose levels and the slopes of the log. dose-response curves did not differ.
Key-words: Diazepam - emulsion - anticonvulsant activity - toxicity intravenous injection.
In previous works (LJUNGBERG& JEPPSSON 1970; JEPPSSON1972a & b; JEPPSSON & LJUNGBERG 1973) pharmacodynamic effects were investigated after parenteral administration of soya bean oil emulsions containing various drugs dissolved in the oil phase. The drugs used were barbituric acids, cyclandelate and nitroglycerin. The results indicated that the emulsion formulations would be suitable vehicles for lipid soluble drugs intended for intravascular administration, since the pharmacological effects were almost equal to those found after a water solution of the sodium salts, which was investigated in the barbituric acid experiments. However, it was not possible in all cases to use water solutions as references, as some drugs have only a limited water solubility. Cyclandelate is an example of a drug slightly soluble in water and therefore it has not been possible to prepare an injection formulation of sufficiently low
ANTICONWLSANT ACTIVITY OF DIAZEPAM
313
toxicity.The cyclandelate emulsion formulation however, seems to be a suitable formulation because the vehicle per se has a low toxicity and cyclandelate in emulsion causes a marked peripheral vasodilatation in perfused cat hind leg in situ and relaxation of guinea pig ileum in uitro (JEPPSSON & LJUNGBERG 1973). An anticonvulsant activity of orally administered diazepam has previously been demonstrated in mice by RANDALLet al. (1961). This effect appeared both after drug induced and electro-shock induced seizures. In the present investigation the anticonvulsant activity of diazepam was observed in mice after intravenous administration. Diazepam is poorly soluble in water and the solvents in the marketed injection formulation Valium" (Hoffmann-La Roche & Co.) are propylene glycol and ethanol. This formulation was compared with an emulsion containing diazepam in the oil phase with regard to toxicity and anticonvulsant activity. The acute intravenous toxicity of two other diazepam injection formulations (apozepam", A. L. and stesolid", Dumex) available in Sweden was also determined.
Materials and Methods Preparations.
The diazepam emulsion was of the following composition: 0.5 g Diazepam (WHO) Soya bean oil 15.0 Acetyl. monoglycerides 5.0 Egg yolk phosphatides 1.2 Glycerol 2.5 Dist. water ad 100.0 ml
The placebo emulsion was of the same composition, but without diazepam. As diazepam solution Valium" inj. was used (ampoules 5 mg/ml, Hoffmann-La Roche & Co.) and as placebo a solution with the same constituents as in Valium" (propylene glycol 41.4%, ethanol 8.06%, benzyl alcohol 1.57%, benzoic acid 0.12% and sodium benzoate 4.88 %). Apozepam" (ampoules 5 mg/ml, A/S Apothekarnes Laboratorium for Specialpreparater) contains phenylcarbinol 1.5 %, glycofurol and ethanol as solvents and stesolid" (Dumex Ltd) phenylcarbinol, propylene glycol and ethanol. Toxicity.
Acute intravenous toxicity (LD50) was estimated in mice of the NMRI-strain (male, 18-22 g) according to the method described in Ph. Nord. (1960). All injections were made into the dorsal tail vein. It was found that the injection speed of Valium" and its placebo, apozepam" and stesolid@had a marked influence on the toxicity. The highest tolerance was found to be below 80 pl/min. so the test was carried out by using an infusion pump injecting the preparations at a constant speed of 62.5 pl/min. Anticonvulsant activity.
PAALZOW (1969) has described a low voltage square wave method for estimation of
R. JEPPSSON AND S. LJUNGBERG
314
anticonvulsant activity, designed to determine repeated seizure thresholds in individual mice with minimal trauma. In the present investigation the voltage of the electrical stimulus was kept constant a t 13.5 V, duration of shock was 400 msec. and frequency 800 p/sec. The electrical stimulator (Grass Mod. S88) was connected to the animals with hypodermic needles applied temporally according to PAALZOW (1969). The test was carried out in two ways. Thus, when various dose levels of diazepam were used (2.5, 5.0, 10.0 and 20.0 mg/kg) the electrical stimulus was applied at certain intervals (64 and 128 min.) after injection. In this way the quanta1 log. dose-response curves were estimated. On the other hand, duration of anticonvulsant activity at a certain dose level was determined by applying the electrical shocks at certain logarithmic intervals up to 256 min. after injection. The decline of effect was estimated after the doses 2.5 and 10.0 mg/kg and the duration of anticonvulsant activity was estimated when 5 0 % of the mice still responded to the drug (DAASO). In all experiments mice of the NMRI-strain (male, 18-24 g) were used. All injections were made into the dorsal tail vein. The preparations tested were diazepam emulsion, placebo emulsion, Valium@inj. and Valium placebo solution.
%
Fig. 1. Anticonvulsant activity (percentage of mice not responding with seizure) of diazepam after intravenous administration of two different injection formulations. Valium@:0 2.5 mg/kg, 0 10.0 mg/kg, A placebo. 2.5 mg/kg, 0 10.0 mg/kg, A placebo. Diazepam emulsion: Untreated.
.......
----
ANTICONVULSANT ACTIVITY OF DIAZEPAM
315
316
R. JEPPSSON AND S. LJUNGBERG
Results Toxicity. The estimated intravenous acute toxicity (LD50) of the formulations are listed in table 1. It was not possible to find the LD50 value of the placebo emulsion because this was tolerated in large amounts, about 1.3 ml/mouse without any toxic signs. Anticonvulsant activity. The frequency of convulsions of untreated mice exposed to the electrical stimulus was 90%. When treated with placebo emulsion, in amounts corresponding to the dose level 10.0 mg/kg of diazepam, the response was almost equal to that of untreated mice (between 80 and 90%). The placebo Valium, administered in the same amounts, had anticonvulsant activity and only 26 % of the animals responded to the stimulus one minute after injection. The anticonvulsant activity declined rapidly and had disappeared 16-32 min. after injection (fig. 1). The anticonvulsant activities of diazepam in emulsion from and of the commercial injection formulation Valium@', at the dose levels 2.5 and 10.0
.,..Solution o
64'
128'
2.5
5.0
10.0
20.0
Dose mg/kg
Fig. 2. Relationship between log. dose and anticonvulsant activity of diazepam in mice after intravenous injection of the two injection formulations Valium@ and diazepam emulsion, estimated 64 min. and 128 min. after the administration.
317
ANTICONVULSANT ACTIVITY OF DIAZEPAM
Table 2. Duration of anticonvulsant activity in 50% of the mice after intravenous administration of Valium@inj. and diazepam in emulsion a t the dose levels 2.5 and 10 mg/kg. Confidence limits are given at P = 0.05. Preparation
DAA5O (min.) at 2.5 mg/kg
DAA5O (min.) at 10 mg/kg
Valium@inj. 5 mg/ml
36.7 (24.4-53.6) n = 206
188 (145-233) n = 110
Diazepam emulsion 5 mg/ml
49.3 (32.9-61.8) n = 160
135 (107-173) n = 110
mg/kg, are shown in fig. 2. At the low dose the emulsion showed a more pronounced time lag compared to that of [email protected] difference between the preparations was not seen at the higher dose level as the anticonvulsant activities were 100% during the first 45 min. The slopes of the decline curves (estimated by probit analysis) did not differ significantly. The duration of anticonvulsant activity in 50% of the mice (DAASO) at the two dose levels used are given in table 2. From these results the EDSO’s were calculated (table 3). The slopes of the regression lines did not differ significantly and neither after 64 nor 128 min. was there any statistically significant difference in anticonvulsant activity between the two preparations. At both 64 and 128 min. the “therapeutic index” of the emulsion formulation was about four times that of Valium@.
Table 3. Acute toxicity (LD50) and dose of Valium@inj. resp. diazepam in emulsion necessary to prevent seizures in 50% of the mice (EDSO) 64 and 128 min. after intravenous injection. “Therapeutic index” is expressed as LD50/ED50. Confidence limits are given at P = 0.05. Preparation
LD5O mg/kg
ED50:64’ mg/kg
LD5O ED50: 64‘
ED50:128’ mdkg
LD50 ED50: 128’
Valium” inj.
83.4 (77.9-8 7.0) n = 50
2.65
31.5
8.06 (6.11-11.4) n = 87
10.4
( 1.80-3.34) n = 105
122.6
6.74 (4.79-9.5 3) n = 80
42.0
Diazepam emulsion
283.3 2.31 (263.5-312.6) (0.88-3.48) n = 60 n = 130
318
R. JEPPSSON AND S. LJUNGBERG
Discussion The low water solubility of diazepam makes it difficult to prepare an injection formulation of low toxicitly. In Valium" injectable diazepam is dissolved with the help of propylene glycol and ethanol but these agents are not pharmacologically inert. In animal experiments propylene glycol has been shown to produce hypnosis, motor incoordination, depression of polysynaptic reflexes and ataxia (CRANKSHAW & RAPER1970). ZAROSLINSKI et al. (1971) have reported an elevation of the convulsion threshold in mice after intraperitioneal administration of propylene glycol. In the present investigation the finding of an anticonvulsant activity of the Valium@placebo formulation, after electrical stimulation of the mouse brain, also points at a pharmacological effect of the solvent mixture itself, and is further supported by the toxicity figure. Earlier, a new way of preparing injection formulations of lipophilic drugs using lipid emulsions as vehicles has been described by LJUNGBERG & JEPPSSON (1970). The toxicity of corresponding placebo emulsions was shown to be low (JEPPSSON 1972a). This is also confirmed in the present investigation. No anticonvulsant activity could be demonstrated with the placebo emulsion. In the present investigation a salient difference in acute toxicity between the commercial injection formulation of diazepam and diazepam emulsion was found. The higher toxicity of the former is most probably due to the toxicity of the formulation per se. The quotient between amounts of placebo Valium" and Valium@inj. given at the level of the LD50 was 1.1 while the same relationship between placebo emulsion and diazepam emulsion could not be estimated because no LD50 of the placebo emulsion could be determined. A rapid distribution of injected diazepam (5 mg/kg) from blood to the brain of the rat and mouse has been reported by MARCUCCI et al. (1968). The highest concentration in the brain was reached within one minute after injection and already 14 sec. after injection of 15 mg/kg. VAN DER KLEIlN (1969) too found a high uptake into mouse brain. Both investigators found a more delayed diazepam distribution to adipose tissue. The rapid onset of anticonvulsant activity registered in the present investigation, at the dose level of 10 mg/kg irrespective of drug formulation, is in accordance with these findings. At the low dose level (2.5 mg/kg) Valium@already showed highest activity at the first reading 4 min. after injection, but diazepam emulsion gave a maximum response after about 25 min. This may be due to a slower distribution to the brain because of a competition between the lipid particles and the CNS for the lipophilic drug. However, since the placebo Valium@per se possessed an anticonvulsant activity, it is not possible to exclude an interaction between the drug and the drug solvent system of the administration formulation. This may also contribute to the more rapid onset of activity noticed after [email protected] between
ANTICONVULSANT ACTIVITY OF DIAZEPAM
319
benzodiazepines and propylene glycol has been reported by CRANKSHAW & RAPER(1970). This was most marked with nitrazepam. At the drug-solvent ratio 5 mg/ml with diazepam, they did not see any interaction when studying loss of righting reflex in mice, but with diminishing drug-solvent ratio such an interaction began to appear. At both dose levels the effect of Valium" and of diazepam emulsion decline in the same manner without any significant difference in anticonvulsant activity (DAASO). Thus, the eventual interaction between diazepam and the solvents could not be demonstrated in the decline of effects, as the curves did not depart from parallellism. The reason for this could be that the effects of the solvent disappeared within a few minutes after administration. In clinical practice LANCDONet al. (1 973) recently reported that Valium@ caused venous pain during injection, especially if smaller veins were used and to produce thrombophlebitis in about 3.5 % of the patients. Such reactions may be correlated with the precipitation of diazepam that occurs when injecting Valium" as reported by JUSKO et al. (1973) or with the solvent system used (MCCLISH1966; LOUISet al. 1967; SHARER & KUTT1971). VON DARDELet al. (1973) compared the incidence of thrombophlebitis in patients injected with enibomal sodium water solution or secobarbital emulsion. The emulsion did not cause any phlebitis while this occurred with the solution in about 30% of the cases. In a preliminary investigation of the diazepam emulsion in patients VON DARDEL (personal communication) found the incidence of phlebitis to be one out of 105 patients. No patients complained about pain at the injection side and the clinical respose did not differ from that of conventional Valium@ therapy. It is important that the solvents of an injectable drug do not interfere with the pharmacon and have no appreceable pharmacological effects per se. The emulsion form seems to fulfil these requirements even if the drug has to pass the blood brain barrier to excert its pharmacological effect. The kinetics concerning this drug diffusion will be dealt with in a forthcoming paper. Acknowledgement The skilful technical assistance of Miss Lena Lutteman and Miss Monica Hellqvist is gratefully acknowledged. We also wish to thank Dr. Ivan HAkansson, Vitrum AB, for preparing the emulsions. One of us, R. J., gratefully acknowledge a grant from "IF :s Stiftelse for Farmaceutisk Forskning".
REFERENCES Crankshaw, D. P. & C. Raper: The effect of solvents on the potency of chlordiazepoxide, diazepam, medazepam and nitrazepam. J . Pharm. Pharmacol. 1970, 23, 313-321. von Dardel, O., C. Mebius & T. Mossberg: A new application form to prevent thrombo-
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phlebitis in intravenous barbituric anaesthesia. XIth Congress of the Scand. SOC.of Anesthesiologists, Reykjavik 1973. Jeppsson, R.: Effects of barbituric acids using an emulsion form intravenously. Acta Pharm. Suec. 1972a, 9, 81-90. Jeppsson, R.: Effects of barbituric acids using an emulsion form intraperitoneally and subcutaneously. Acta Pharm. Suec. 1972b, 9, 199-206. Jeppsson, R. & S. Ljungberg : Intraarterial administration of emulsion formulations containing cyclandelate and nitroglycerin. Acta Pharm. Suec. 1973, 10, 129-140. Jusko, W. J., M. Gretch & R. Gasset: Precipitation of diazepam from intravenous preparations. J. Amer. Med. Assoc. 1973, 223, 176. van der Kleijn, E.: Kinetics of distribution and metabolism of diazepam and chlordiazepoxide in mice. Arch. int. Pharmacodyn. 1969, 178, 193-215. Langdon, D. E., J. R. Harlan & R. L. Bailey: Thrombophlebitis with diazepam used intravenously. J. Amer. Med. Assoc. 1973, 223, 184-185. Ljungberg, S. & R. Jeppsson: Intravenous administration of lipid soluble drugs. Acta Pharm. Suec. 1970,7, 435440. Louis, S., H. Kutt & F. McDowell: The cardiocirculatory changes caused by intravenous dilantin and its solvent. Amer. Heart J. 1967, 74, 523-529. Marcucci, F., A. Guaitani, J. Kvetina, E. Mussini & S. Garattini: Species differences in diazepam metabolism and anticonvulsant effect. Eur. J. Pharmacol. 1968, 4,467-470. McClish, A. : Diazepam as an intravenous induction agent for general anesthesia. Can. Anes. SOC.J. 1966,13, 562-575. Paalzow, L.: An electrical method for estimation of anticonvulsant activity in mice, I. Acta Pharm. Suec. 1969, 6 , 163-176. Pharmacopoea Nordica Vol. 1V, Ed. Suec., Apotekarsocietetens forlag, Stockholm 1960, pp. 31. Randall, L. O., G. A. Heise, W. Schallek, R. E. Bagdon, R. Banziger, A. Boris, R. A. Moe & W. B. Abrams: Pharmacological and clinical studies on Valium@ a new psychoterapeutic agent of the benzodiazepine class. Curr. Therap. Res. 1961, 3 , 409425. Sharer, L. & H. Kutt: Intrapenous administration of diazepam. Arch. Neurol. 1971, 24, 169-175. Zaroslinski, J. F., R. K. Browne & L. H. Possley: Propylene glycol as a drug solvent in pharmacologic studies. Toxicol. Appl. Pharmacol. 1971,19, 573-578.
From the Department of Pharmacology, Faculty of Pharmacy, University of Uppsala, Box 573, S-751 23 Uppsala, Sweden
Anticonvulsant Activity in Mice of Diazepam in an Emulsion Formulation for Intravenous Administration BY Roland Jeppsson and Stellan Ljungberg (Received October 23, 1974; Accepted November 4, 1974)
Abstract: The anticonvulsant activity of diazepam has been investigated in mice using an electrical low voltage square wave technique. Diazepam was administered intravenously using two injection formulations, Valium@inj. (Roche) and diazepam emulsion. In the latter formulation the lipophilic drug was dissolved in soya bean oil which was then emulsified to an o/w emulsion. A great difference was found in acute intravenous toxicity (LD50) between the two diazepam formulations. The higher toxicity of Valium" inj. could probably be due to the solvents used in this formulation. The anticonvulsant activity was about equal for the two preparations. Thus, no difference was found in duration of anticonvulsant activity at the two dose levels and the slopes of the log. dose-response curves did not differ.
Key-words: Diazepam - emulsion - anticonvulsant activity - toxicity intravenous injection.
In previous works (LJUNGBERG& JEPPSSON 1970; JEPPSSON1972a & b; JEPPSSON & LJUNGBERG 1973) pharmacodynamic effects were investigated after parenteral administration of soya bean oil emulsions containing various drugs dissolved in the oil phase. The drugs used were barbituric acids, cyclandelate and nitroglycerin. The results indicated that the emulsion formulations would be suitable vehicles for lipid soluble drugs intended for intravascular administration, since the pharmacological effects were almost equal to those found after a water solution of the sodium salts, which was investigated in the barbituric acid experiments. However, it was not possible in all cases to use water solutions as references, as some drugs have only a limited water solubility. Cyclandelate is an example of a drug slightly soluble in water and therefore it has not been possible to prepare an injection formulation of sufficiently low
ANTICONWLSANT ACTIVITY OF DIAZEPAM
313
toxicity.The cyclandelate emulsion formulation however, seems to be a suitable formulation because the vehicle per se has a low toxicity and cyclandelate in emulsion causes a marked peripheral vasodilatation in perfused cat hind leg in situ and relaxation of guinea pig ileum in uitro (JEPPSSON & LJUNGBERG 1973). An anticonvulsant activity of orally administered diazepam has previously been demonstrated in mice by RANDALLet al. (1961). This effect appeared both after drug induced and electro-shock induced seizures. In the present investigation the anticonvulsant activity of diazepam was observed in mice after intravenous administration. Diazepam is poorly soluble in water and the solvents in the marketed injection formulation Valium" (Hoffmann-La Roche & Co.) are propylene glycol and ethanol. This formulation was compared with an emulsion containing diazepam in the oil phase with regard to toxicity and anticonvulsant activity. The acute intravenous toxicity of two other diazepam injection formulations (apozepam", A. L. and stesolid", Dumex) available in Sweden was also determined.
Materials and Methods Preparations.
The diazepam emulsion was of the following composition: 0.5 g Diazepam (WHO) Soya bean oil 15.0 Acetyl. monoglycerides 5.0 Egg yolk phosphatides 1.2 Glycerol 2.5 Dist. water ad 100.0 ml
The placebo emulsion was of the same composition, but without diazepam. As diazepam solution Valium" inj. was used (ampoules 5 mg/ml, Hoffmann-La Roche & Co.) and as placebo a solution with the same constituents as in Valium" (propylene glycol 41.4%, ethanol 8.06%, benzyl alcohol 1.57%, benzoic acid 0.12% and sodium benzoate 4.88 %). Apozepam" (ampoules 5 mg/ml, A/S Apothekarnes Laboratorium for Specialpreparater) contains phenylcarbinol 1.5 %, glycofurol and ethanol as solvents and stesolid" (Dumex Ltd) phenylcarbinol, propylene glycol and ethanol. Toxicity.
Acute intravenous toxicity (LD50) was estimated in mice of the NMRI-strain (male, 18-22 g) according to the method described in Ph. Nord. (1960). All injections were made into the dorsal tail vein. It was found that the injection speed of Valium" and its placebo, apozepam" and stesolid@had a marked influence on the toxicity. The highest tolerance was found to be below 80 pl/min. so the test was carried out by using an infusion pump injecting the preparations at a constant speed of 62.5 pl/min. Anticonvulsant activity.
PAALZOW (1969) has described a low voltage square wave method for estimation of
R. JEPPSSON AND S. LJUNGBERG
314
anticonvulsant activity, designed to determine repeated seizure thresholds in individual mice with minimal trauma. In the present investigation the voltage of the electrical stimulus was kept constant a t 13.5 V, duration of shock was 400 msec. and frequency 800 p/sec. The electrical stimulator (Grass Mod. S88) was connected to the animals with hypodermic needles applied temporally according to PAALZOW (1969). The test was carried out in two ways. Thus, when various dose levels of diazepam were used (2.5, 5.0, 10.0 and 20.0 mg/kg) the electrical stimulus was applied at certain intervals (64 and 128 min.) after injection. In this way the quanta1 log. dose-response curves were estimated. On the other hand, duration of anticonvulsant activity at a certain dose level was determined by applying the electrical shocks at certain logarithmic intervals up to 256 min. after injection. The decline of effect was estimated after the doses 2.5 and 10.0 mg/kg and the duration of anticonvulsant activity was estimated when 5 0 % of the mice still responded to the drug (DAASO). In all experiments mice of the NMRI-strain (male, 18-24 g) were used. All injections were made into the dorsal tail vein. The preparations tested were diazepam emulsion, placebo emulsion, Valium@inj. and Valium placebo solution.
%
Fig. 1. Anticonvulsant activity (percentage of mice not responding with seizure) of diazepam after intravenous administration of two different injection formulations. Valium@:0 2.5 mg/kg, 0 10.0 mg/kg, A placebo. 2.5 mg/kg, 0 10.0 mg/kg, A placebo. Diazepam emulsion: Untreated.
.......
----
ANTICONVULSANT ACTIVITY OF DIAZEPAM
315
316
R. JEPPSSON AND S. LJUNGBERG
Results Toxicity. The estimated intravenous acute toxicity (LD50) of the formulations are listed in table 1. It was not possible to find the LD50 value of the placebo emulsion because this was tolerated in large amounts, about 1.3 ml/mouse without any toxic signs. Anticonvulsant activity. The frequency of convulsions of untreated mice exposed to the electrical stimulus was 90%. When treated with placebo emulsion, in amounts corresponding to the dose level 10.0 mg/kg of diazepam, the response was almost equal to that of untreated mice (between 80 and 90%). The placebo Valium, administered in the same amounts, had anticonvulsant activity and only 26 % of the animals responded to the stimulus one minute after injection. The anticonvulsant activity declined rapidly and had disappeared 16-32 min. after injection (fig. 1). The anticonvulsant activities of diazepam in emulsion from and of the commercial injection formulation Valium@', at the dose levels 2.5 and 10.0
.,..Solution o
64'
128'
2.5
5.0
10.0
20.0
Dose mg/kg
Fig. 2. Relationship between log. dose and anticonvulsant activity of diazepam in mice after intravenous injection of the two injection formulations Valium@ and diazepam emulsion, estimated 64 min. and 128 min. after the administration.
317
ANTICONVULSANT ACTIVITY OF DIAZEPAM
Table 2. Duration of anticonvulsant activity in 50% of the mice after intravenous administration of Valium@inj. and diazepam in emulsion a t the dose levels 2.5 and 10 mg/kg. Confidence limits are given at P = 0.05. Preparation
DAA5O (min.) at 2.5 mg/kg
DAA5O (min.) at 10 mg/kg
Valium@inj. 5 mg/ml
36.7 (24.4-53.6) n = 206
188 (145-233) n = 110
Diazepam emulsion 5 mg/ml
49.3 (32.9-61.8) n = 160
135 (107-173) n = 110
mg/kg, are shown in fig. 2. At the low dose the emulsion showed a more pronounced time lag compared to that of [email protected] difference between the preparations was not seen at the higher dose level as the anticonvulsant activities were 100% during the first 45 min. The slopes of the decline curves (estimated by probit analysis) did not differ significantly. The duration of anticonvulsant activity in 50% of the mice (DAASO) at the two dose levels used are given in table 2. From these results the EDSO’s were calculated (table 3). The slopes of the regression lines did not differ significantly and neither after 64 nor 128 min. was there any statistically significant difference in anticonvulsant activity between the two preparations. At both 64 and 128 min. the “therapeutic index” of the emulsion formulation was about four times that of Valium@.
Table 3. Acute toxicity (LD50) and dose of Valium@inj. resp. diazepam in emulsion necessary to prevent seizures in 50% of the mice (EDSO) 64 and 128 min. after intravenous injection. “Therapeutic index” is expressed as LD50/ED50. Confidence limits are given at P = 0.05. Preparation
LD5O mg/kg
ED50:64’ mg/kg
LD5O ED50: 64‘
ED50:128’ mdkg
LD50 ED50: 128’
Valium” inj.
83.4 (77.9-8 7.0) n = 50
2.65
31.5
8.06 (6.11-11.4) n = 87
10.4
( 1.80-3.34) n = 105
122.6
6.74 (4.79-9.5 3) n = 80
42.0
Diazepam emulsion
283.3 2.31 (263.5-312.6) (0.88-3.48) n = 60 n = 130
318
R. JEPPSSON AND S. LJUNGBERG
Discussion The low water solubility of diazepam makes it difficult to prepare an injection formulation of low toxicitly. In Valium" injectable diazepam is dissolved with the help of propylene glycol and ethanol but these agents are not pharmacologically inert. In animal experiments propylene glycol has been shown to produce hypnosis, motor incoordination, depression of polysynaptic reflexes and ataxia (CRANKSHAW & RAPER1970). ZAROSLINSKI et al. (1971) have reported an elevation of the convulsion threshold in mice after intraperitioneal administration of propylene glycol. In the present investigation the finding of an anticonvulsant activity of the Valium@placebo formulation, after electrical stimulation of the mouse brain, also points at a pharmacological effect of the solvent mixture itself, and is further supported by the toxicity figure. Earlier, a new way of preparing injection formulations of lipophilic drugs using lipid emulsions as vehicles has been described by LJUNGBERG & JEPPSSON (1970). The toxicity of corresponding placebo emulsions was shown to be low (JEPPSSON 1972a). This is also confirmed in the present investigation. No anticonvulsant activity could be demonstrated with the placebo emulsion. In the present investigation a salient difference in acute toxicity between the commercial injection formulation of diazepam and diazepam emulsion was found. The higher toxicity of the former is most probably due to the toxicity of the formulation per se. The quotient between amounts of placebo Valium" and Valium@inj. given at the level of the LD50 was 1.1 while the same relationship between placebo emulsion and diazepam emulsion could not be estimated because no LD50 of the placebo emulsion could be determined. A rapid distribution of injected diazepam (5 mg/kg) from blood to the brain of the rat and mouse has been reported by MARCUCCI et al. (1968). The highest concentration in the brain was reached within one minute after injection and already 14 sec. after injection of 15 mg/kg. VAN DER KLEIlN (1969) too found a high uptake into mouse brain. Both investigators found a more delayed diazepam distribution to adipose tissue. The rapid onset of anticonvulsant activity registered in the present investigation, at the dose level of 10 mg/kg irrespective of drug formulation, is in accordance with these findings. At the low dose level (2.5 mg/kg) Valium@already showed highest activity at the first reading 4 min. after injection, but diazepam emulsion gave a maximum response after about 25 min. This may be due to a slower distribution to the brain because of a competition between the lipid particles and the CNS for the lipophilic drug. However, since the placebo Valium@per se possessed an anticonvulsant activity, it is not possible to exclude an interaction between the drug and the drug solvent system of the administration formulation. This may also contribute to the more rapid onset of activity noticed after [email protected] between
ANTICONVULSANT ACTIVITY OF DIAZEPAM
319
benzodiazepines and propylene glycol has been reported by CRANKSHAW & RAPER(1970). This was most marked with nitrazepam. At the drug-solvent ratio 5 mg/ml with diazepam, they did not see any interaction when studying loss of righting reflex in mice, but with diminishing drug-solvent ratio such an interaction began to appear. At both dose levels the effect of Valium" and of diazepam emulsion decline in the same manner without any significant difference in anticonvulsant activity (DAASO). Thus, the eventual interaction between diazepam and the solvents could not be demonstrated in the decline of effects, as the curves did not depart from parallellism. The reason for this could be that the effects of the solvent disappeared within a few minutes after administration. In clinical practice LANCDONet al. (1 973) recently reported that Valium@ caused venous pain during injection, especially if smaller veins were used and to produce thrombophlebitis in about 3.5 % of the patients. Such reactions may be correlated with the precipitation of diazepam that occurs when injecting Valium" as reported by JUSKO et al. (1973) or with the solvent system used (MCCLISH1966; LOUISet al. 1967; SHARER & KUTT1971). VON DARDELet al. (1973) compared the incidence of thrombophlebitis in patients injected with enibomal sodium water solution or secobarbital emulsion. The emulsion did not cause any phlebitis while this occurred with the solution in about 30% of the cases. In a preliminary investigation of the diazepam emulsion in patients VON DARDEL (personal communication) found the incidence of phlebitis to be one out of 105 patients. No patients complained about pain at the injection side and the clinical respose did not differ from that of conventional Valium@ therapy. It is important that the solvents of an injectable drug do not interfere with the pharmacon and have no appreceable pharmacological effects per se. The emulsion form seems to fulfil these requirements even if the drug has to pass the blood brain barrier to excert its pharmacological effect. The kinetics concerning this drug diffusion will be dealt with in a forthcoming paper. Acknowledgement The skilful technical assistance of Miss Lena Lutteman and Miss Monica Hellqvist is gratefully acknowledged. We also wish to thank Dr. Ivan HAkansson, Vitrum AB, for preparing the emulsions. One of us, R. J., gratefully acknowledge a grant from "IF :s Stiftelse for Farmaceutisk Forskning".
REFERENCES Crankshaw, D. P. & C. Raper: The effect of solvents on the potency of chlordiazepoxide, diazepam, medazepam and nitrazepam. J . Pharm. Pharmacol. 1970, 23, 313-321. von Dardel, O., C. Mebius & T. Mossberg: A new application form to prevent thrombo-
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phlebitis in intravenous barbituric anaesthesia. XIth Congress of the Scand. SOC.of Anesthesiologists, Reykjavik 1973. Jeppsson, R.: Effects of barbituric acids using an emulsion form intravenously. Acta Pharm. Suec. 1972a, 9, 81-90. Jeppsson, R.: Effects of barbituric acids using an emulsion form intraperitoneally and subcutaneously. Acta Pharm. Suec. 1972b, 9, 199-206. Jeppsson, R. & S. Ljungberg : Intraarterial administration of emulsion formulations containing cyclandelate and nitroglycerin. Acta Pharm. Suec. 1973, 10, 129-140. Jusko, W. J., M. Gretch & R. Gasset: Precipitation of diazepam from intravenous preparations. J. Amer. Med. Assoc. 1973, 223, 176. van der Kleijn, E.: Kinetics of distribution and metabolism of diazepam and chlordiazepoxide in mice. Arch. int. Pharmacodyn. 1969, 178, 193-215. Langdon, D. E., J. R. Harlan & R. L. Bailey: Thrombophlebitis with diazepam used intravenously. J. Amer. Med. Assoc. 1973, 223, 184-185. Ljungberg, S. & R. Jeppsson: Intravenous administration of lipid soluble drugs. Acta Pharm. Suec. 1970,7, 435440. Louis, S., H. Kutt & F. McDowell: The cardiocirculatory changes caused by intravenous dilantin and its solvent. Amer. Heart J. 1967, 74, 523-529. Marcucci, F., A. Guaitani, J. Kvetina, E. Mussini & S. Garattini: Species differences in diazepam metabolism and anticonvulsant effect. Eur. J. Pharmacol. 1968, 4,467-470. McClish, A. : Diazepam as an intravenous induction agent for general anesthesia. Can. Anes. SOC.J. 1966,13, 562-575. Paalzow, L.: An electrical method for estimation of anticonvulsant activity in mice, I. Acta Pharm. Suec. 1969, 6 , 163-176. Pharmacopoea Nordica Vol. 1V, Ed. Suec., Apotekarsocietetens forlag, Stockholm 1960, pp. 31. Randall, L. O., G. A. Heise, W. Schallek, R. E. Bagdon, R. Banziger, A. Boris, R. A. Moe & W. B. Abrams: Pharmacological and clinical studies on Valium@ a new psychoterapeutic agent of the benzodiazepine class. Curr. Therap. Res. 1961, 3 , 409425. Sharer, L. & H. Kutt: Intrapenous administration of diazepam. Arch. Neurol. 1971, 24, 169-175. Zaroslinski, J. F., R. K. Browne & L. H. Possley: Propylene glycol as a drug solvent in pharmacologic studies. Toxicol. Appl. Pharmacol. 1971,19, 573-578.
