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In vivo uptake of valproic acid into brain
Brain Research, 240 (1982) 195-198
195
Elsevier Biomedical Press
In vivo uptake of valproic acid into brain E. J. HAMMOND, R. J. PERCHALSKI, H. J. VILLARREAL and B. J. WILDER Neurology and Medical Research Service, Veterans Administration Medical Center, Gainesville, FL 32602 (U.S.A.)
(Accepted February 4th, 1982) Key words: valproic acid - - dipropylacetate - - anticonvulsants - - brain uptake
The pharmacokinetics of valproic acid (VPA) penetration into the central nervous system of cats were studied. VPA levels in cortical gray matter and plasma were measured at timed intervals after rapid intravenous drug infusion. Brain uptake of the drug was maximal at 1 min postinfusion and decayed rapidly with a mean elimination half-life of 41 min. After a rapid distribution phase, plasma VPA levelsremained stable for 90 rain. The brain :plasma ratio was maximal at 1 min and also declined rapidly. The volume of distribution was 0.125 l/kg. The small volume of distribution, low brain :plasma ratios and rapid clearance from brain indicate that VPA is not significantlybound in cerebral cortex after a single dose. Valproic acid (di-n-propylacetic acid, VPA) is an anticonvulsant extremely effective in generalized seizure disorders 2~. Numerous mechanisms of action have been proposedf, 7. Although the plasma kinetics of VPA have been reported for animals 17,19 and mann, 16,1s, little is known about the central nervous system (CNS) penetration of this drug. Here we report analysis of this relationship in a manner similar to that which we have reported for other antiepileptic drugs 14. Six adult cats (5.5-7.5 kg) in good health were anesthetized with a mixture of halothane, nitrous oxide and oxygen in an open circuit, non-rebreathing system. The anesthetic mixture was administered by means of a tracheal cannula. The animals breathed freely. The cats were placed in head holder and the cerebral cortex was exposed. The dura was left intact until just before the taking of tissue sampies. Sodium valproate (60 mg/kg dissolved in saline) was linearly infused (3 min) into the inferior vena cava by a catheter inserted into the right femoral vein. Simultaneous samples of cerebral cortex and plasma were collected at intervals of 1, 3, 6, 10, 15, 30, 45, 60 and 90 min after the end of the drug infusion. The plasma samples were taken from a catheter in the left femoral vein. Plasma in the dead space of the catheter was cleared by use of a 3way stopcock before samples were taken. Cortical 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
samples were weighed immediately and averaged 150 mg. Adjacent areas of cortex were covered with cotton soaked in warm saline. No cortical edema was observed during the course of the experiments. Plasma and brain levels of VPA were determined by gas-liquid chromatography. The details of the plasma VPA assay have been described previously 1. The weighed brain tissue was homogenized in 1 ml saline. After addition of the internal standard (cyclopentyl acetic acid) and 6 ~ perchloric acid, the sample was extracted with chloroform. Before evaporation of the organic phase a small volume of amylacetate was added to prevent loss of valproic acid. The sample was taken from the small volume of amylacetate. Plasma would be expected to comprise less than 1 ~ of the brain volumeS, 15. Kinetic parameters were determined by fitting straight lines to the points and applying standard formulae for calculationsS,1L The rapid distribution phase (a-phase) brain half-life was 6.58 min q- 6.49 (mean ± S.D. ; range 1.4--13.7 min) and the elimination phase (fl-phase) brain half-life of the drug was 41.1 min ± 8.3 (range 34.6--55.4 min). The volume of distribution was calculated to be 0.125 ± 0.018 liter/kg (range: 0.07-0.195). The brain clearance was 1.47 d: 0.13 ml/kg/min (range: 1.3-1.6). The plasma levels for a given cat were seen to remain rather stable for the duration of the experiment, whereas
196
"L 10
-~.3J,-~
Pu--..a
]
\
~-
B
'
~ m
X • ....
•
MEAN
<
6O
MINUTES AFTER INFUSION
C
7O
O0
~0
CALLN CH/FIC FAKTO
Fig. 1. Pharmacokineties of valproic acid after single intravenous dose (60 mg/kg) in the cat. A: brain valproic acid leveltime profile. B: brain:plasma ratios. C: plasma level-time profile. In A, B and C, data from individual cats are plotted to
'
the brain level of VPA decreased rapidly to low levels by 90 min (Fig. 1); at 1 rain postinfusion the main brain :plasma ratio was 0.72, which declined to 0.11 by 90 rain. Although the same intravenous dosage was given to all cats, the plasma concentrations varied somewhat (Fig. 1); at 30 min postinfusion the highest plasma level was 150 #g/ml. In the cats that maintained plasma levels greater than 60 /~g/ml, the brain levels remained relatively more stable than in cats in which the plasma levels were lower. The halothane anesthetic used in this study is highly lipophilic and may increase permeability of the blood-brain barrier to non-polar molecules, such as VPA. However, although the rates of passage may be increased, the effect should be equivalent for drug uptake and release. An equilibrium will still be attained that would allow valid conclusions to be reached concerning brain binding. The dissimilarity of these drugs should preclude any metabolic interaction. Although the kinetics of VPA have been described by a one compartment model a7,1~, rapid intravenous drug administration and short sampling intervals allowed for the determination of a biexpotential curve in this study. Recent detailed measurements in man have also suggested an open two compartment modell6,1s. The low apparent volume of distribution (0.125 liter/kg) is very similar to that found in humans 9Aa. This indicates that VPA is not significantly bound to brain and suggests that high plasma levels are necessary to achieve appreciable brain levels. Valproic acid is highly protein bound 1 and only the free plasma fraction is available for brain penetration. The kinetics of distribution of VPA across the plasma-cerebrospinal fluid (CSF) interface have recently been reported~,l°; Frey and Loscher 1° suggested that VPA is transported into and out of the CSF by the monocarboxylic acid transport system. In this study plasma levels of less than 60 #g/ml were accompanied by extremely low brain levels (Fig. 1). In humans clinical effectiveness of VPA is generally only realized with relatively high plasma levels, above 55/~g/ml. show relationship between plasma level and brain level for individual cats.
197 The brain :plasma ratio found for VPA is significantly different than that found for other antiepileptic drugs; Ramsay et al. 14 found ratios of greater than 1.0 for phenytoin, phenobarbital and diazepam in cats, and Vadja et al. 21 found ratios of 0.75 for phenytoin and 0.59 for phenobarbital in epileptic patients. The relatively high clearance and short half-life in brain, and low brain :plasma ratio as compared with other antiepileptic drugs raises questions as to what VPA's mechanism of action might be. Using radiolabeled VPA, Goldberg and Todoroff 4 were unable to demonstrate binding to any fraction of homogenates of whole mouse brain. A differential regional distribution has, however, been reported by some investigators: Schobben and colleagues showed VPA to be localized in white matter tracts in the cerebellum and occipital lobe la, and Ciesielski et al. 2 found relatively low concentrations of VPA in the cortex but reported high levels in the striatum and fornix. The dose of valproate used in this study (60 mg/kg) resulted in cortical valproate concen-
Supported by the Medical Research Service of the Veterans Administration and the Epilepsy Research Foundation of Florida. H.J.V. was supported by A N U I E S , Mexico City, Mexico. The authors thank Mitchell Thomas, Shirley V. Dunbar and Millie Walden for technical and secretarial assistance.
1 Bruni, J., Wilder, B. J., Willmore, L. J., Perchalski, R. J. and Villarreal, H. J., Pharmacokinetics of steady state valproic acid in epileptic patients, Clin. Pharmacol. Ther., 24 (1978) 324-332. 2 Ciesielski, L., Maitre, M., Cash, C. and Mandel, P., Regional distribution in brain and effect on cerebral mitochondrial respiration of the anticonvulsive drug n-dipropylacetate, Biochem. Pharmacol., 24 (1975) 1055-1058. 3 Frey, H. H. and Loscher, W., Distribution of valproate across the interface between blood and cerebrospinal fluid, Neuropharmacology, 17 (1978) 637-642. 4 Goldberg, M. A. and Todoroff, T., Brain binding of anticonvulsants: carbamazel~ine and valproic acid, Neurology, 30 (1980) 826-832. ' 5 Goodman, L. S. and Gilman, A. (Eds.), The Pharmacologic Basis of Therapeutics, Macmillan, New York, 1975, pp. 19-21. 6 Hackman, J. C., Grayson, V. and Davidoff, R. A., The presynaptic effects of valproic acid in the isolated from spinal cord, Brain Research, 220 (1981) 269-285. 7 Hammond, E. J., Wilder, B. J. and Bruni, J., Central actions of valproic acid in man and in experimental models of epilepsy, Life Sci., 29 (1981) 2561-2574. 8 Hossmann, K. A. and Zimmerman, V., Resuscitation of the monkey brain after 1 h of complete ischemia. I. Physiologic and morphological observations, Brain Research, 81 (1974) 59-74. 9 Klotz, V. and Antonin, K. H., Pharmacokinetics and bioavailability of sodium valproate, Clin. PharmacoL Ther., 21 (1977) 736-743.
10 Levy, R. H., CSF and plasma pharmacokinetics: relationship to mechanisms of action as exemplified by valproic acid in monkey. In J. S. Lockard and A. A. Ward (Eds.), Epilepsy: A Window to Brain Mechanisms, Raven Press, New York, 1980, pp. 191-200. 11 Lockard, J. and Levy, R. H., Valproic acid: reversibly acting drug? Epilepsia, 17 (1976) 477-479. 12 Melmon, K. L. and MereUi, H. F. (Eds.), ClinicalPharmacology, Macmillan, New York, 1972, pp. 39-48. 13 Pellegrini, A., Gloor, P. and Sherwin, A. L., Effect of valproate sodium on generalized penicillin epilepsy in the cat, Epilepsia, 19 (1978) 351-360. 14 Ramsay, R. E., Hammond, E. J., Perchalski, R. J. and Wilder, B. J., Brain uptake of phenytoin, phenobarbital and diazepam, Arch. Neurol., 36 (1979) 535-539. 15 Rosomoff, H. L., Method for simultaneous quantitative estimation of intracranial contents, J. appl. Physiol., 16 (1961) 395-396. 16 Schapel, G. J., Beran, R. G., Docshke, C. J., O'Reilly, W. J., et al., Pharmacokinetics of sodium valproate in epileptic patients: prediction of maintenance dosage by single dose study, Europ. J. Clin. Pharmacol., 17 (1980) 71-77. 17 Schobben, F. and Van der Kleijn, E., Pharmacokinetics of distribution and elimination of sodium di-N-propylacetate in mouse and dog, Pharm. Weekbl., 109 (1974) 33-41. 18 Schobben, F., Van der Kleijn, E. and Gabreels, F. J. M., Pharmacokinetics of di-N-propylacetate in epileptic patients, Europ. J. Clin. Pharmacol., 8 (1975) 97-105. 19 Schobben, F. and Van der Kleijn, E., Elimination and
trations averaging 40 pg/g, after the initial distribution phase. Vajda et al. 20 also found relatively low concentrations 6-27 pg/g) of valproate in human brain samples taken during surgery. Valproic acid is structurally different from the traditional antiepileptic drugs and has dissimilar pharmacokinetic parameters, suggesting a novel mode of action. Reports of anticonvulsant effect long after the drug is withdrawn 11,13, when contrasted with the apparent lack of binding, suggests a slow process, either the accumulation of an active metabolite or alteration of some metabolic process in the brain. It may be that VPA incorporation into the brain has a certain latency and/or is a rate controlled multi-step process.
198 distribution of 2-N-propyl pentonate in monkeys, Pharm. WeekbL, 112 (1977) 345-347. 20 Vajda, F. J. E., Donnan, G. A., Phillips, J. and Bladin, P. F., Human brain, plasma and cerebrospinal fluid concentration of sodium valproate after 72 hours of therapy, Neurology, 31 (1981) 486-487. 21 Vajda, F., Williams, F. M., Davidson, S., Falconer, M. A.
and Breckenridge, A., Human brain, cerebrospinal fluid, and plasma concentrations of diphenylhydantoin and phenobarbital, Clin. Pharmacol. Ther., 15 (1974)597-603. 22 Wilder, B. J. and Bruni, J., Seizure Disorders: A Pharmacological Approach to Treatment, Raven Press, New York, 1981, pp. 83-93.
195
Elsevier Biomedical Press
In vivo uptake of valproic acid into brain E. J. HAMMOND, R. J. PERCHALSKI, H. J. VILLARREAL and B. J. WILDER Neurology and Medical Research Service, Veterans Administration Medical Center, Gainesville, FL 32602 (U.S.A.)
(Accepted February 4th, 1982) Key words: valproic acid - - dipropylacetate - - anticonvulsants - - brain uptake
The pharmacokinetics of valproic acid (VPA) penetration into the central nervous system of cats were studied. VPA levels in cortical gray matter and plasma were measured at timed intervals after rapid intravenous drug infusion. Brain uptake of the drug was maximal at 1 min postinfusion and decayed rapidly with a mean elimination half-life of 41 min. After a rapid distribution phase, plasma VPA levelsremained stable for 90 rain. The brain :plasma ratio was maximal at 1 min and also declined rapidly. The volume of distribution was 0.125 l/kg. The small volume of distribution, low brain :plasma ratios and rapid clearance from brain indicate that VPA is not significantlybound in cerebral cortex after a single dose. Valproic acid (di-n-propylacetic acid, VPA) is an anticonvulsant extremely effective in generalized seizure disorders 2~. Numerous mechanisms of action have been proposedf, 7. Although the plasma kinetics of VPA have been reported for animals 17,19 and mann, 16,1s, little is known about the central nervous system (CNS) penetration of this drug. Here we report analysis of this relationship in a manner similar to that which we have reported for other antiepileptic drugs 14. Six adult cats (5.5-7.5 kg) in good health were anesthetized with a mixture of halothane, nitrous oxide and oxygen in an open circuit, non-rebreathing system. The anesthetic mixture was administered by means of a tracheal cannula. The animals breathed freely. The cats were placed in head holder and the cerebral cortex was exposed. The dura was left intact until just before the taking of tissue sampies. Sodium valproate (60 mg/kg dissolved in saline) was linearly infused (3 min) into the inferior vena cava by a catheter inserted into the right femoral vein. Simultaneous samples of cerebral cortex and plasma were collected at intervals of 1, 3, 6, 10, 15, 30, 45, 60 and 90 min after the end of the drug infusion. The plasma samples were taken from a catheter in the left femoral vein. Plasma in the dead space of the catheter was cleared by use of a 3way stopcock before samples were taken. Cortical 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
samples were weighed immediately and averaged 150 mg. Adjacent areas of cortex were covered with cotton soaked in warm saline. No cortical edema was observed during the course of the experiments. Plasma and brain levels of VPA were determined by gas-liquid chromatography. The details of the plasma VPA assay have been described previously 1. The weighed brain tissue was homogenized in 1 ml saline. After addition of the internal standard (cyclopentyl acetic acid) and 6 ~ perchloric acid, the sample was extracted with chloroform. Before evaporation of the organic phase a small volume of amylacetate was added to prevent loss of valproic acid. The sample was taken from the small volume of amylacetate. Plasma would be expected to comprise less than 1 ~ of the brain volumeS, 15. Kinetic parameters were determined by fitting straight lines to the points and applying standard formulae for calculationsS,1L The rapid distribution phase (a-phase) brain half-life was 6.58 min q- 6.49 (mean ± S.D. ; range 1.4--13.7 min) and the elimination phase (fl-phase) brain half-life of the drug was 41.1 min ± 8.3 (range 34.6--55.4 min). The volume of distribution was calculated to be 0.125 ± 0.018 liter/kg (range: 0.07-0.195). The brain clearance was 1.47 d: 0.13 ml/kg/min (range: 1.3-1.6). The plasma levels for a given cat were seen to remain rather stable for the duration of the experiment, whereas
196
"L 10
-~.3J,-~
Pu--..a
]
\
~-
B
'
~ m
X • ....
•
MEAN
<
6O
MINUTES AFTER INFUSION
C
7O
O0
~0
CALLN CH/FIC FAKTO
Fig. 1. Pharmacokineties of valproic acid after single intravenous dose (60 mg/kg) in the cat. A: brain valproic acid leveltime profile. B: brain:plasma ratios. C: plasma level-time profile. In A, B and C, data from individual cats are plotted to
'
the brain level of VPA decreased rapidly to low levels by 90 min (Fig. 1); at 1 rain postinfusion the main brain :plasma ratio was 0.72, which declined to 0.11 by 90 rain. Although the same intravenous dosage was given to all cats, the plasma concentrations varied somewhat (Fig. 1); at 30 min postinfusion the highest plasma level was 150 #g/ml. In the cats that maintained plasma levels greater than 60 /~g/ml, the brain levels remained relatively more stable than in cats in which the plasma levels were lower. The halothane anesthetic used in this study is highly lipophilic and may increase permeability of the blood-brain barrier to non-polar molecules, such as VPA. However, although the rates of passage may be increased, the effect should be equivalent for drug uptake and release. An equilibrium will still be attained that would allow valid conclusions to be reached concerning brain binding. The dissimilarity of these drugs should preclude any metabolic interaction. Although the kinetics of VPA have been described by a one compartment model a7,1~, rapid intravenous drug administration and short sampling intervals allowed for the determination of a biexpotential curve in this study. Recent detailed measurements in man have also suggested an open two compartment modell6,1s. The low apparent volume of distribution (0.125 liter/kg) is very similar to that found in humans 9Aa. This indicates that VPA is not significantly bound to brain and suggests that high plasma levels are necessary to achieve appreciable brain levels. Valproic acid is highly protein bound 1 and only the free plasma fraction is available for brain penetration. The kinetics of distribution of VPA across the plasma-cerebrospinal fluid (CSF) interface have recently been reported~,l°; Frey and Loscher 1° suggested that VPA is transported into and out of the CSF by the monocarboxylic acid transport system. In this study plasma levels of less than 60 #g/ml were accompanied by extremely low brain levels (Fig. 1). In humans clinical effectiveness of VPA is generally only realized with relatively high plasma levels, above 55/~g/ml. show relationship between plasma level and brain level for individual cats.
197 The brain :plasma ratio found for VPA is significantly different than that found for other antiepileptic drugs; Ramsay et al. 14 found ratios of greater than 1.0 for phenytoin, phenobarbital and diazepam in cats, and Vadja et al. 21 found ratios of 0.75 for phenytoin and 0.59 for phenobarbital in epileptic patients. The relatively high clearance and short half-life in brain, and low brain :plasma ratio as compared with other antiepileptic drugs raises questions as to what VPA's mechanism of action might be. Using radiolabeled VPA, Goldberg and Todoroff 4 were unable to demonstrate binding to any fraction of homogenates of whole mouse brain. A differential regional distribution has, however, been reported by some investigators: Schobben and colleagues showed VPA to be localized in white matter tracts in the cerebellum and occipital lobe la, and Ciesielski et al. 2 found relatively low concentrations of VPA in the cortex but reported high levels in the striatum and fornix. The dose of valproate used in this study (60 mg/kg) resulted in cortical valproate concen-
Supported by the Medical Research Service of the Veterans Administration and the Epilepsy Research Foundation of Florida. H.J.V. was supported by A N U I E S , Mexico City, Mexico. The authors thank Mitchell Thomas, Shirley V. Dunbar and Millie Walden for technical and secretarial assistance.
1 Bruni, J., Wilder, B. J., Willmore, L. J., Perchalski, R. J. and Villarreal, H. J., Pharmacokinetics of steady state valproic acid in epileptic patients, Clin. Pharmacol. Ther., 24 (1978) 324-332. 2 Ciesielski, L., Maitre, M., Cash, C. and Mandel, P., Regional distribution in brain and effect on cerebral mitochondrial respiration of the anticonvulsive drug n-dipropylacetate, Biochem. Pharmacol., 24 (1975) 1055-1058. 3 Frey, H. H. and Loscher, W., Distribution of valproate across the interface between blood and cerebrospinal fluid, Neuropharmacology, 17 (1978) 637-642. 4 Goldberg, M. A. and Todoroff, T., Brain binding of anticonvulsants: carbamazel~ine and valproic acid, Neurology, 30 (1980) 826-832. ' 5 Goodman, L. S. and Gilman, A. (Eds.), The Pharmacologic Basis of Therapeutics, Macmillan, New York, 1975, pp. 19-21. 6 Hackman, J. C., Grayson, V. and Davidoff, R. A., The presynaptic effects of valproic acid in the isolated from spinal cord, Brain Research, 220 (1981) 269-285. 7 Hammond, E. J., Wilder, B. J. and Bruni, J., Central actions of valproic acid in man and in experimental models of epilepsy, Life Sci., 29 (1981) 2561-2574. 8 Hossmann, K. A. and Zimmerman, V., Resuscitation of the monkey brain after 1 h of complete ischemia. I. Physiologic and morphological observations, Brain Research, 81 (1974) 59-74. 9 Klotz, V. and Antonin, K. H., Pharmacokinetics and bioavailability of sodium valproate, Clin. PharmacoL Ther., 21 (1977) 736-743.
10 Levy, R. H., CSF and plasma pharmacokinetics: relationship to mechanisms of action as exemplified by valproic acid in monkey. In J. S. Lockard and A. A. Ward (Eds.), Epilepsy: A Window to Brain Mechanisms, Raven Press, New York, 1980, pp. 191-200. 11 Lockard, J. and Levy, R. H., Valproic acid: reversibly acting drug? Epilepsia, 17 (1976) 477-479. 12 Melmon, K. L. and MereUi, H. F. (Eds.), ClinicalPharmacology, Macmillan, New York, 1972, pp. 39-48. 13 Pellegrini, A., Gloor, P. and Sherwin, A. L., Effect of valproate sodium on generalized penicillin epilepsy in the cat, Epilepsia, 19 (1978) 351-360. 14 Ramsay, R. E., Hammond, E. J., Perchalski, R. J. and Wilder, B. J., Brain uptake of phenytoin, phenobarbital and diazepam, Arch. Neurol., 36 (1979) 535-539. 15 Rosomoff, H. L., Method for simultaneous quantitative estimation of intracranial contents, J. appl. Physiol., 16 (1961) 395-396. 16 Schapel, G. J., Beran, R. G., Docshke, C. J., O'Reilly, W. J., et al., Pharmacokinetics of sodium valproate in epileptic patients: prediction of maintenance dosage by single dose study, Europ. J. Clin. Pharmacol., 17 (1980) 71-77. 17 Schobben, F. and Van der Kleijn, E., Pharmacokinetics of distribution and elimination of sodium di-N-propylacetate in mouse and dog, Pharm. Weekbl., 109 (1974) 33-41. 18 Schobben, F., Van der Kleijn, E. and Gabreels, F. J. M., Pharmacokinetics of di-N-propylacetate in epileptic patients, Europ. J. Clin. Pharmacol., 8 (1975) 97-105. 19 Schobben, F. and Van der Kleijn, E., Elimination and
trations averaging 40 pg/g, after the initial distribution phase. Vajda et al. 20 also found relatively low concentrations 6-27 pg/g) of valproate in human brain samples taken during surgery. Valproic acid is structurally different from the traditional antiepileptic drugs and has dissimilar pharmacokinetic parameters, suggesting a novel mode of action. Reports of anticonvulsant effect long after the drug is withdrawn 11,13, when contrasted with the apparent lack of binding, suggests a slow process, either the accumulation of an active metabolite or alteration of some metabolic process in the brain. It may be that VPA incorporation into the brain has a certain latency and/or is a rate controlled multi-step process.
198 distribution of 2-N-propyl pentonate in monkeys, Pharm. WeekbL, 112 (1977) 345-347. 20 Vajda, F. J. E., Donnan, G. A., Phillips, J. and Bladin, P. F., Human brain, plasma and cerebrospinal fluid concentration of sodium valproate after 72 hours of therapy, Neurology, 31 (1981) 486-487. 21 Vajda, F., Williams, F. M., Davidson, S., Falconer, M. A.
and Breckenridge, A., Human brain, cerebrospinal fluid, and plasma concentrations of diphenylhydantoin and phenobarbital, Clin. Pharmacol. Ther., 15 (1974)597-603. 22 Wilder, B. J. and Bruni, J., Seizure Disorders: A Pharmacological Approach to Treatment, Raven Press, New York, 1981, pp. 83-93.