Psychopharmacology (1991) 103 : 384-387 003331589100041B
Psychopharmacology © Springer-Verlag 1991
Stability and pharmacokinetics of flumazenil in the rat Jaap W. Mandema, Josy M. Gubbens-Stibbe, and Meindert Danhof Center for Bio-Pharmaceutical Sciences, Division of Pharmacology, University of Leiden, P.O. Box 9503, NL-2300 RA Leiden, The Netherlands Received April 5, 1990 / Final version August 1, 1990
Abstract. The pharmacokinetics of flumazenil in the rat were determined after 2.5 mg/kg intravenous and 25 mg/kg oral administration. Following intravenous administration flumazenil was rapidly eliminated with an extremely short terminal half-life ( m e a n ± SE, n = 8) of 8.3±0.3 min due to a large total blood clearance o f 147 4- 7 ml/kg/min combined with a relatively small volume of distribution at steady-state of 1.33 4-0.07 1/kg. After oral administration flumazenil was rapidly absorbed; however, the bioavailability was low (28 + 4 % ) and variable. Flumazenil was found to be unstable in rat blood in vitro and disappeared with a half-life (mean 4- SE, n = 5) of 8.3 4- 1 rain and 31 4- 4 rain at body and room temperature, respectively. The blood samples were stabilized by addition o f sodium fluoride (NaF) and cooling to 0 ° C. The samples had to be stored at - 35 ° C when analyzed at later times. Presumably esterases in rat blood are responsible for the observed instability. A sensitive H P L C assay to measure flumazenil concentrations in small blood samples is also described.
Key words: Flumazenil - Pharmacokinetics
ing on the test and dosage of flumazenil used, the route of administration and the time point of effect measurement (for reviews see File and Pellow 1986; Brogden and Goa 1988). During the past 10 years flumazenil has been extensively used as a pharmacological tool in studies on the behaviourial pharmacology o f benzodiazepines in both laboratory animals and humans. For the better interpretation and design of pharmacological studies conducted with ftumazenil, insight into the pharmacokinetic properties o f the drug is essential. In rats, to our knowledge, only one study has appeared in the literature so far reporting an elimination half-life of 16 min from rat brain after intraperitoneal administration of 10 mg/kg (Lister et al. 1984). However, in this study no measurable plasma concentrations could be detected. The purpose of the present investigation was to study the pharmacokinetics of flumazenil in rats after intravenous and oral administration. Prior to these studies, the stability o f flumazenil in rat blood in vitro was determined.
Rat - Sta-
bility - H P L C assay
Materials and methods Flumazenil (Ro 15-1788, Fig. 1) was the first benzodiazepine derivative combining a high affinity for the central benzodiazepine receptor with no intrinsic pharmacological activity (Hunkeler et al. 1981). Flumazenil was shown to selectively antagonize central effects of benzodiazepines in a variety of tests (Hunkeler et al. 198I ; P o l c et al. 1981 ; Bonetti et al, 1982; Heafely et al. 1985), presumably by competitive inhibition of benzodiazepines binding to the central benzodiazepine receptor (M6hler and Richards 198t). Since the original reports that flumazenil had no intrinsic activity, evidence has been gathered that flumazenil nevertheless possesses some agonistic or even inverse agonistic activity dependOffprint requests to: M. Danhof
Animals. Male SPF rats from Wistar descent weighing 200--250 g were used throughout the study. The animals were kept on a commercially available diet (Standard Laboratory Rat, Mouse and
0
// \ 0 CH~ Fig. 1. Chemical structure offlumazenil (ethyl 8-fluoro-5,6-dihydro5-methyl-6-oxo-4H-imidazo-[1,5-a][l,4]benzodiazepine-3-carboxylate)
385 Hamster Diets, RMH-TM, Hope Farms, Woerden, The Netherlands).
Stability offlumazenil in vitro. The stability of flumazenil in rat whole blood in vitro was studied at body temperature (38° C) and room temperature (23° C). Fresh rat blood was obtained from five animals by means of a puncture of the aorta immediately before the experiment. Flumazenil was added to the rat blood at a concentration of 5 rag/1. Blood from each rat was divided into two portions which were kept at 38° C and 23° C respectively, and continuously gently shaken. At fixed time points samples of 100 gl were taken and immediately assayed as described in the drug analysis section. The stabilizing effect of addition of 0.5 ml 0.42% NaF solution to 100 gl blood samples containing flumazenil was tested after storing the solutions for 4 h at 0° C and 37° C and 2 weeks at - 35° C at two different concentrations of flumazenil. Pharmacokinetics. To determine the pharmacokinetics of flumazenil in the rat, indwelling cannulas were implanted in the right jugular vein and right femoral artery under light ether anaesthesia 1 day before the experiments. After the operation the animals were housed individually. From the night before the experiment onwards the animals received no food but had free access to water. The pharmacokinetics of flumazenil were determined in eight rats after intravenous bolus administration of 2.5 mg/kg and in four rats after oral administration of 25 mg/kg. For intravenous administration flumazenil was dissolved in a 40% solution of PEG 4000 (Brocacef, Maarsen, The Netherlands) in 0.9% saline. For oral administration flumazenil was suspended in 0.9 % saline using tween 20 (2 drops per 10 ml). A total volume of 0.5 ml was administered both orally and intravenously. The dosages selected are within the dose range normally used in studies on the pharmacological activity of flumazenil in the rat. Arterial blood samples (100 gl) were frequently drawn and immediately diluted with 0.5 ml ice-cold 0.42% sodium fluoride (NaF) solution in water to inhibit esterase activity and kept on ice until the end of the experiment. Immediately after the experiment, the samples were stored - 3 5 ° C until the time of analysis, which was always within 2 weeks after the experiment. Drug analysb. A sensitive assay was developed to measure flumazenil concentrations in small blood samples (100 gl whole blood) by HPLC with UV detection. A solution of 60 ng nitrazepam (internal standard) in 20 pl methanol was added to the mixture of 100 gl whole blood and 0.5 mt NaF solution. Subsequently, the samples were further diluted with 0.5 ml borate buffer (0.2 M, pH = 9.0). The mixture was extracted with 5 ml dichloromethanepentane (1 : 1) for 30 s on a vortex mixer. After centrifugation the organic layer was separated and evaporated to dryness under reduced pressure. The residue was reconstituted with 200 gl mobile phase and 150 pl was injected into the chromatographic system. The chromatographic system consisted of a M-45 solvent pump, a WISP 710B automatic sample injector, a 30 cm gBondapak C18 steel column and a Lambda-Max Model 481 LC Spectrophotometer set at 250 nm (all of Waters Associates, Milford, MA, USA). The mobile phase consisted of a mixture of 0.01 M citrate/phosphate buffer, pH = 4.0 and methanol (Baker, Deventer, The Netherlands) in a ratio 45/55 with a flow rate of 1.2 ml/min. Retention times were about 4.5 rain and 9 mJn for flumazenil and nitrazepam, respectively. Data processing was performed using a Shimadzu C-R3A integrator. The coefficients of variation for identical samples (within day variation) were (n=5): 3.6% at 20 ng/ml, 2.0% at 50 ng/ml, 0.91% at 500 ng/ml and 0.70% at 5000 ng/ml. The between-day coefficients of variation at these concentrations were, respectively, 5.8, 4.5, 1.6 and 0.22%. The detection limit was about 10 ng/ml. Data analysis. The concentration time profiles of flumazenil after intravenous bolus and oral administration and in vitro decomposition experiments were described using a multi-exponential equation :
Cb = £ Aie air (Eq. 1) i=l
where Cb is the blood concentration of flumazenil, A i and cq are, respectively, the coefficients and exponents and n is the number of exponentials. In case of oral administration the curve was forced through the origin. The equations were fitted to the data using the nonlinear least squares regression program Siphar (Siphar pharmacokinetic modelling software package, SIMED S.A., Creteil, France). Different exponential models were investigated and the most suitable model was chosen according to standard criteria. Basic pharmacokinetic parameters as area under the curve (AUC), whole blood clearance (Clb), volume of distribution in steady state (Vds~) and terminal half-life (twa%) were calculated for the individual rats according to standard procedures using the coefficients and exponents of the fitted functions (Gibatdi and Perrier 1982). The bioavailability (F) after oral administration was calculated from the averaged AUCs after intravenous and oral administration• Statistical comparisons were made using a Student's t-test. The probability level was set at 5% (P
Psychopharmacology © Springer-Verlag 1991
Stability and pharmacokinetics of flumazenil in the rat Jaap W. Mandema, Josy M. Gubbens-Stibbe, and Meindert Danhof Center for Bio-Pharmaceutical Sciences, Division of Pharmacology, University of Leiden, P.O. Box 9503, NL-2300 RA Leiden, The Netherlands Received April 5, 1990 / Final version August 1, 1990
Abstract. The pharmacokinetics of flumazenil in the rat were determined after 2.5 mg/kg intravenous and 25 mg/kg oral administration. Following intravenous administration flumazenil was rapidly eliminated with an extremely short terminal half-life ( m e a n ± SE, n = 8) of 8.3±0.3 min due to a large total blood clearance o f 147 4- 7 ml/kg/min combined with a relatively small volume of distribution at steady-state of 1.33 4-0.07 1/kg. After oral administration flumazenil was rapidly absorbed; however, the bioavailability was low (28 + 4 % ) and variable. Flumazenil was found to be unstable in rat blood in vitro and disappeared with a half-life (mean 4- SE, n = 5) of 8.3 4- 1 rain and 31 4- 4 rain at body and room temperature, respectively. The blood samples were stabilized by addition o f sodium fluoride (NaF) and cooling to 0 ° C. The samples had to be stored at - 35 ° C when analyzed at later times. Presumably esterases in rat blood are responsible for the observed instability. A sensitive H P L C assay to measure flumazenil concentrations in small blood samples is also described.
Key words: Flumazenil - Pharmacokinetics
ing on the test and dosage of flumazenil used, the route of administration and the time point of effect measurement (for reviews see File and Pellow 1986; Brogden and Goa 1988). During the past 10 years flumazenil has been extensively used as a pharmacological tool in studies on the behaviourial pharmacology o f benzodiazepines in both laboratory animals and humans. For the better interpretation and design of pharmacological studies conducted with ftumazenil, insight into the pharmacokinetic properties o f the drug is essential. In rats, to our knowledge, only one study has appeared in the literature so far reporting an elimination half-life of 16 min from rat brain after intraperitoneal administration of 10 mg/kg (Lister et al. 1984). However, in this study no measurable plasma concentrations could be detected. The purpose of the present investigation was to study the pharmacokinetics of flumazenil in rats after intravenous and oral administration. Prior to these studies, the stability o f flumazenil in rat blood in vitro was determined.
Rat - Sta-
bility - H P L C assay
Materials and methods Flumazenil (Ro 15-1788, Fig. 1) was the first benzodiazepine derivative combining a high affinity for the central benzodiazepine receptor with no intrinsic pharmacological activity (Hunkeler et al. 1981). Flumazenil was shown to selectively antagonize central effects of benzodiazepines in a variety of tests (Hunkeler et al. 198I ; P o l c et al. 1981 ; Bonetti et al, 1982; Heafely et al. 1985), presumably by competitive inhibition of benzodiazepines binding to the central benzodiazepine receptor (M6hler and Richards 198t). Since the original reports that flumazenil had no intrinsic activity, evidence has been gathered that flumazenil nevertheless possesses some agonistic or even inverse agonistic activity dependOffprint requests to: M. Danhof
Animals. Male SPF rats from Wistar descent weighing 200--250 g were used throughout the study. The animals were kept on a commercially available diet (Standard Laboratory Rat, Mouse and
0
// \ 0 CH~ Fig. 1. Chemical structure offlumazenil (ethyl 8-fluoro-5,6-dihydro5-methyl-6-oxo-4H-imidazo-[1,5-a][l,4]benzodiazepine-3-carboxylate)
385 Hamster Diets, RMH-TM, Hope Farms, Woerden, The Netherlands).
Stability offlumazenil in vitro. The stability of flumazenil in rat whole blood in vitro was studied at body temperature (38° C) and room temperature (23° C). Fresh rat blood was obtained from five animals by means of a puncture of the aorta immediately before the experiment. Flumazenil was added to the rat blood at a concentration of 5 rag/1. Blood from each rat was divided into two portions which were kept at 38° C and 23° C respectively, and continuously gently shaken. At fixed time points samples of 100 gl were taken and immediately assayed as described in the drug analysis section. The stabilizing effect of addition of 0.5 ml 0.42% NaF solution to 100 gl blood samples containing flumazenil was tested after storing the solutions for 4 h at 0° C and 37° C and 2 weeks at - 35° C at two different concentrations of flumazenil. Pharmacokinetics. To determine the pharmacokinetics of flumazenil in the rat, indwelling cannulas were implanted in the right jugular vein and right femoral artery under light ether anaesthesia 1 day before the experiments. After the operation the animals were housed individually. From the night before the experiment onwards the animals received no food but had free access to water. The pharmacokinetics of flumazenil were determined in eight rats after intravenous bolus administration of 2.5 mg/kg and in four rats after oral administration of 25 mg/kg. For intravenous administration flumazenil was dissolved in a 40% solution of PEG 4000 (Brocacef, Maarsen, The Netherlands) in 0.9% saline. For oral administration flumazenil was suspended in 0.9 % saline using tween 20 (2 drops per 10 ml). A total volume of 0.5 ml was administered both orally and intravenously. The dosages selected are within the dose range normally used in studies on the pharmacological activity of flumazenil in the rat. Arterial blood samples (100 gl) were frequently drawn and immediately diluted with 0.5 ml ice-cold 0.42% sodium fluoride (NaF) solution in water to inhibit esterase activity and kept on ice until the end of the experiment. Immediately after the experiment, the samples were stored - 3 5 ° C until the time of analysis, which was always within 2 weeks after the experiment. Drug analysb. A sensitive assay was developed to measure flumazenil concentrations in small blood samples (100 gl whole blood) by HPLC with UV detection. A solution of 60 ng nitrazepam (internal standard) in 20 pl methanol was added to the mixture of 100 gl whole blood and 0.5 mt NaF solution. Subsequently, the samples were further diluted with 0.5 ml borate buffer (0.2 M, pH = 9.0). The mixture was extracted with 5 ml dichloromethanepentane (1 : 1) for 30 s on a vortex mixer. After centrifugation the organic layer was separated and evaporated to dryness under reduced pressure. The residue was reconstituted with 200 gl mobile phase and 150 pl was injected into the chromatographic system. The chromatographic system consisted of a M-45 solvent pump, a WISP 710B automatic sample injector, a 30 cm gBondapak C18 steel column and a Lambda-Max Model 481 LC Spectrophotometer set at 250 nm (all of Waters Associates, Milford, MA, USA). The mobile phase consisted of a mixture of 0.01 M citrate/phosphate buffer, pH = 4.0 and methanol (Baker, Deventer, The Netherlands) in a ratio 45/55 with a flow rate of 1.2 ml/min. Retention times were about 4.5 rain and 9 mJn for flumazenil and nitrazepam, respectively. Data processing was performed using a Shimadzu C-R3A integrator. The coefficients of variation for identical samples (within day variation) were (n=5): 3.6% at 20 ng/ml, 2.0% at 50 ng/ml, 0.91% at 500 ng/ml and 0.70% at 5000 ng/ml. The between-day coefficients of variation at these concentrations were, respectively, 5.8, 4.5, 1.6 and 0.22%. The detection limit was about 10 ng/ml. Data analysis. The concentration time profiles of flumazenil after intravenous bolus and oral administration and in vitro decomposition experiments were described using a multi-exponential equation :
Cb = £ Aie air (Eq. 1) i=l
where Cb is the blood concentration of flumazenil, A i and cq are, respectively, the coefficients and exponents and n is the number of exponentials. In case of oral administration the curve was forced through the origin. The equations were fitted to the data using the nonlinear least squares regression program Siphar (Siphar pharmacokinetic modelling software package, SIMED S.A., Creteil, France). Different exponential models were investigated and the most suitable model was chosen according to standard criteria. Basic pharmacokinetic parameters as area under the curve (AUC), whole blood clearance (Clb), volume of distribution in steady state (Vds~) and terminal half-life (twa%) were calculated for the individual rats according to standard procedures using the coefficients and exponents of the fitted functions (Gibatdi and Perrier 1982). The bioavailability (F) after oral administration was calculated from the averaged AUCs after intravenous and oral administration• Statistical comparisons were made using a Student's t-test. The probability level was set at 5% (P
