- Email: [email protected]
Acute anticonvulsant activity of structural analogues of valproic acid and changes in brain GABA and aspartate content
Life Sciences, Vol. 32, pp. 2023-2031 Printed in the U.S.A.
Pergamon Press
ACUTE ANTICONVULSANT ACTIVITY OF STRUCTURAL ANALOGUES OF VALPROIC ACID AND CHANGES IN BRAIN GABA AND ASPARTATE CONTENT Astrid G. Chapman 1, B r i a n S. Meldrum 2 and E t i e n n e Mendes 3 Department of Neurology I, Rayne Institute, King's College Hospital Medical School, 123 Coldharbour ~ane, London, SE5 9NU. Department of Neurology-, Institute of Psychiatry, De Crespigny Park, London, ~E5 8AF, U.K. Sanofi Recherche , 195 Route d'Espagne, 31300 Toulouse Cedex, France. (Received in final form January 31, 1983) Summary Ten analogues of valproic acid (substituted butyric, pentanoic and hexanoic acids) were tested for anticonvulsant activity against audiogenic seizures in DBA/2 mice. There is a consistent correlation between the structure of these branched-chain fatty acids and their anticonvulsant potency, the larger molecules being the m o ~ a c t i v e . There is also a strong correlation between the anticonvulsant potency of these compounds and their ability to reduce cerebral aspartate levels. Cerebral GABA levels are elevated by most, but not all, of the actively anticonvulsant valproate analogues. The anticonvulsant activity of valproic acid (2-propyl-pentanoic acid) or its sodium or magnesium salts, has been known since 1963. Valproate is currently used to treat several syndromes of epilepsy (see I-2) but its mechanisms of action remains essentially unknown (3). The principal inhibitory neurotransmitter in the mammalian brain is GABA (4-aminobutyric acid). Benzodiazepines and barbiturates are thought to owe at least part of their anticonvulsant action to enhancement of the postsynapic inhibitory action of GABA (4-6). Brain GABA content is increased after the acute administration of valproate to rodents (7-9). In vitro valproate inhibits all of the three enzymes involved in the further metabolism of GABA (GABAtransaminase, succinic semialdehyde dehydrogenase, and aldehyde reductase) but its in vivo effect on GABA metabolism is uncertain (see 10). Nevertheless the biochemical data have been interpreted in terms of valproate enhancing GABAergic inhibition by making more transmitter available for synaptic release (11). The acute administration of valproate also decreases brain aspartate content (9,12-13). The possible significance of this effect has been recently emphasized by the discovery of the anticonvulsant properties of amino acid analogues that preferentially block excitation due to N-methyl-D-aspartate or aspartic acid Cas opposed to excitation due to kainic acid, quisqualic acid or glutamate) (14-15). The effects of valproate on GABA and aspartate appear to be independent. Changes in concentration of GABA and aspartate 0024-3205/83/172023-09503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
2024
Valproate
Analogues,
Brain G A B A and ASP
Vol.
32, No.
17, 1983
show a different gross regional distribution in the rat brain (9) and a different s u b c e l l u l a r d i s t r i b u t i o n in brain homogenates (16). We have examined the acute a n t i c o n v u l s a n t action, of a series of valproate analogues (i.e. b r a n c h e d - c h a i n fatty acids with carbon chain lengths of 4-7) in DBA/2 mice showing s o u n d - i n d u c e d seizures that are age dependent (17). For comparative purposes we have also included 3 straight chain fatty acids. A time point of 30 min was selected for drug evaluation as the effect of valproic acid against a u d i o g e n i c seizures is fully established at this time (13, 18), and the sedative and other side effects of the s t r a i g h t - c h a i n and branchedchain analogues show a similar time course to those of valproic acid. We have also measured forebrain aspartate and GABA concentrations after drug treatments to assess the correlation of such changes with a n t i c o n v u l s a n t action. Methods All experiments were performed Psychiatry from LAC stock.
in DBA/2 mice
bred at the Institute
of
For e v a l u a t i o n of the behavioural and a n t i c o n v u l s a n t drug a c t i o n mice of either sex, 21-26 days old (body weights 6-13 g) were injected with olive oil (B.P. Boots Ltd.) 0.1 ml, or with the test compound, 0.5-4 mmoles/kg, dissolved in 0.1 ml olive oil. Thirty min after drug a d m i n i s t r a t i o n rectal temperature was measured and the animal placed under a perspex dome (diameter 58 cm) for observation of motor activity. Stimulation by an electric bell (Friedland chimes, 3 inch diameter, p r o d u c i n g 109 dB at mouse level) was applied for 60 sec or until tonic extension occurred. Time to onset of each phase of the seizure response was recorded. A seizure response (SR) score was calculated for each animal on the basis of the maximal response:0 = none; I = wild running; 2 = clonus; 3 = tonic extension; 4 = respiratory arrest. Later, sequential, phases are indicated by higher scores. Later phases were never seen in the absence of the p r e c e d i n g (lower score) phase. For groups of 8-10 mice receiving one dose of drug, differences in the occurrence of seizure phases (compared with carrier treated litter mate mice, run at the same time) were evaluated with Fisher's exact test. For each phase of the seizure response, the EDeN for s u p p r e s s i o n of that phase was calculated g r a p h i c a l l y (using 4 l o g a r i t ~ i c a l l y augmented doses). The drug dose reducing the SR score by 50% was also calculated. For biochemical studies mice were injected i n t r a p e r i t o n e a l l y with 2 m m o l e s / k g of each acid in olive oil, 0.1 ml, 30 min prior to d e c a p i t a t i o n into liquid nitrogen. The heads were kept in liquid nitrogen until the forebrains were chisled out on the same day with liquid N_ irrigation and extracted with 5 volumes of 0.4 N HCI04, I mM EDTA and s u b s e q u e n t l y neutralized with a KOHNaHCO 3 mixture. Aspartate levels in the extract were determined e n z y m a t i c a l l y using the malate dehydrogenase (MDH) linked assay. The concentrations in the assay mix (I ml) were: Tris. HC1, pH 8.0:60 mM, ~ - k e t o g l u t a r a t e : O . 2 mM, NADH:O.I mM, MDH:2.5 ~ g / m l . Thirty ~l brain extract or standard aspartate solution (1050 nmoles) was used per ml of assay mix. The reactions were started with the addition of g l u t a m a t e - o x a l o a c e t a t e amino-transferase (GOT) and the decrease in absorbance at 340 mm was determined s p e c t r o p h o t o m e t r i c a l l y . GABA levels were determined f l u o r o m e t r i c a l l y using the GABAase assay. The concentrations in the final reaction mix (2.5 ml) were: Na pyrophosphate buffer, pH 8.6: 60 mM, NADP+:IO mM, m e r c a p t o e t h a n o l : 6 mM, ~ - k e t o g l u t a r a t e : 2 mM. The reaction mixture contained 0.25 ml brain extract or standard GABA (20-150 nmoles/ 2.5 ml) solution, and the reaction was initiated by the addition of
Vol. 32, No. 17, 1983
Valproate Analogues,
Brain GABA and ASP
2025
-ketoglutarateo The analogues listed in Table I were either obtained from commercial sources and repurified by distillation, or synthesised in the laboratories of Sanofi Recherche, Toulouse, by malonic synthesis (for methyl-2-pentanoic acid) or ~-alkylation of the corresponding carboxylic acid as described by Pfeffer et al (19). The purity of all the analogues was determined by gas chromatography, infrared and NMR characteristics, and by elementary analysis. Results A typical audiogenic seizure in DBA/2 mice consists of a wild running phase (latency I-6 sec), a clonic phase (latency 5-15 sec), and tonic extension (latency 10-35 sec), frequently followed by respiratory arrest (latency 25-50 sec). Figure I shows the percentage of DBA/2 mice (n = 8-10 per dose) undergoing the sound-induced wild running and clonic phases following the intraperitoneal administration of a series of valproate analogues (0.5-4 mmoles/kg; 30 min). Similar response curves, indicating slightly lower effective doses of the analogues, were obtained for the tonic extension and respiratory arrest phases of the seizure (data not shown). The anticonvulsant effect of valproic acid is shown by curve 7 in Figure I. Four of the valproate analogues, 2-ethyl-hexanoic acid, 2-propyl-hexanoic acid, 2-propylheptano~c acid, and 2-butyl-hexanoic acid (curves 8-11) are more potent anticonvulsants than valproate. The short, straight-chain fatty acids, butyric, pentanoic, and hexanoic acid, as well as 2-ethyl-pentanoic acid (curves I-4) have little or no anticonvulsant properties. 2-Methyl-pentanoic acid and 2-ethyl-butyric acid display intermediate anticonvulsant activity (curves 5-6).
WILD
RUNNING
CLONIC PHASE
!
1,2,3
100
80
7
1
s
80
uJ
u) 60
60
Z O
o_ 40
6
4(]
IJJ
Oc
20
20
o
0
!
!
!
!
|
05
I
2
4
0.5
mmol/kg
|
!
I
1
2
4
mmol / kg
FIG. I Effect of valproate analogues (0.5 - 4 mmoles/kg, i.p. 30 min prior to test) on the initial phases of the sound induced seizure response. Fatty acids indicated are: I. hexanoic acid; 2. pentanoic acid; 3. 2-ethyl-pentanoic acid; 4. butyric acid; 5. 2-methyl-pentanoic acid; 6. 2-ethyl-butyric acid; 7. ~alproic acid; 8. 2-ethyl-hexanoic acid; 9. 2-propyl-hexanoic acid; 10. 2-propylheptanoic acid; 11. 2-butyl-hexanoic acid.
2026
Valproate
Analogues,
Brain GABA and ASP
Vol.
32, No.
17,
1983
The inactive or w e a k l y a n t i c o n v u l s a n t compounds (I-6 in Fig. I) have little or no effect on the behaviour of the mice in doses up to 2 mmoles/kg. At mmoles/kg these compounds produce a slight sedation, piloerection, and peritoneal irritation, as well as a slight ata×ia in some cases. Valproic acid produces no behavioural effects at doses of 0.5 and I mmole/kg, a slight ataxia at 2 mmcles/kg, and heavy sedation and ataxia at 4 mmoles/kg. The four potent a n t i c o n v u l s a n t analogues (8-11 in Fig. I) produce no behavioural effects at 0.5 mmoles/kg, and slight sedation with fairly normal exploration at I mmole/kg. At 2 mmole/kg 2 - e t h y l - h e x a n o i c acid produces slight sedation and no ata×ia, whereas 2 - p r o p y l - h e x a n o i c acid produces moderate sedation and slight ataxia. The mice receiving 2 m m o l e s / k g 2 - b u t y l - h e × a n o i c acid or 2p r o p y l - h e p t a n o i c acid were comatose, and 2 mice died in the latter group. I00
so
R/CHCOOH
I
LLI 1 0 0 U') < -r"
o
o
[1_ 0
~_
50
HCOOH
0 _.J 0
7 0
--
0._ 100 O3 LLI
o
o
L
5o
I
R:
ox
H
|
I
f
q%
R"
HCOOH
|
gH,,
FIG. 2 Relationship between chain length and a n t i c o n v u l s a n t potency of valproate analogues. The effect of I m m o l e / k g of valproate analogues (i.p. 30 min) on the clonic phase of audiogenic seizures in DBA/2 mice. The filled symbol denotes valproate. R = Alpha s u b s t i t u t i o n for three formulae illustrated.
Vol. 32, No. 17, 1983
Valproate
Analogues, Brain GABA and ASP
2027
The administration of valproate and all the valproate analogues resulted in a dose-dependent reduction in the rectal temperature of the mice: Control value: 37.2~0.06 °, 0.5 mmoles/kg: 36.930.1 ° , I mmole/kg: 36.430.1 °, 2 mmoles/ kg: 35.3~O.6 °, and 4 mmoles/kg: 34.7~O.2 °. The reductions were similar for the active and the inactive analogues, and they were not severe enough to affect the audiogenic seizure response (20). Figure 2 shows the relationship between the structure of the valproate analogues and their anticonvulsant potency expressed as clonic seizure response observed following the administration of I mmole/kg of these analogues. The anticonvulsant potency of these branched-chain fatty acids is proportional to the chain length of the side group for all the three branched fatty acid series: substituted hexanoic, pentanoic, and butyric acid. The anticonvulsant potencies of the valproate analogues, expressed as ED (mM) against the clonic phase of the audiogenic seizures and against the 50 overall seizure response (SR) score are listed in Table I, along with the forebrain levels of GABA and aspartate determined 30 min after the administration of 2 mmoles/kg of these compounds. TABLE I (A) ED50 for Suppression of Audiogenic Seizures in DBA/2 Mice by Valproate Analogues and (B) the Effect of Administration of these Analogues (2 mmoles/kg i.p., 30 min) on the Aspartate and GABA Levels in Forebrain.
(A) Fatty Acid
Anticonv. WR
Control Hexanoic Pentanoic Butyric 2-Ethyl-Pentanoic 2-Methyl-Pentanoic 2-Ethyl-Butyric Valproic 2-Propyl-Heptanoic 2-Propyl-Hexanoic 2-Butyl-Hexanoic 2-Ethyl-Hexanoic
7.0 8.0 4.0 2.8 1.4 0.72 0.85 0.75 0.90
Potency
(B) (ED5omM)
Clonic
SR
10.0 5.8 5.6 4.8 4.0 2.6 1.25 0.72 0.68 0.68 0.66
8.0 6.0 5.0 5.0 2.7 2.4 1.1 0.7 0.7 0.7 0.5
Brain Conc. Aspartate 2.7730.13 2.86-0.14 2.2330.18 * 3.0630.13 2.2130.15" 2.3030.11" 2.1030.11" 1.3030.12"** 1.2530.06 *** 1.3230.08"** 1.2730.06 *** 0.9830.11"**
(~moles/g) GABA 1.4530.06 1.4030.04 2.06-0.14" 1.5330.08 2.0930.05 *** 1.5730.08 1.5830.10 2.7730.20 *** 2.0630.09*** 1.8330.08 ** 2.1930.05 *** 2.9430.14"**
Valproate analogues are listed in order of increasing + overall anticonvulsant efficacy. Amino acid values are expressed as mean - SEM; statistical difference from control values (Student's t-test): *p < 0.05; ** p < 0.01; *** p < 0.001. SR : Seizure response score (see methods). The administration of valproic acid and the four most potent anticonvulsant analogues, 2-propyl-heptanoic acid, 2-propyl-hexanoic acid, 2-butyl-hexanoic acid, and 2-ethyl-hexanoic acid results in 52-65% reductions in the aspartate levels and 26-103% increases in the GABA levels in the forebrain.
2028
Valproate Analogues,
Brain GABA and ASP
Vol. 32, No. 17, 1983
The relationship between the anticonvulsant potency of the valproate analogues and their effect on the forebrain amino acid levels is illustrated graphically in Figure 3.
{ 0
E I < n7
<
o ,
,
,
,
,
,
,
,
,
,
,
'
lb
, , f /
,
{
i 0
E-j 0_ U~
<_
6
'
'
'
¢
'
;s
CdNrROL
EDso,mM. CLONIC PHASE FIG. 3 Cerebral aspartate and GABA levels following the administration of valproate analogues (2 mmoles/kg i.p., 30 min) to DBA/2 mice, versus the potency of these analogues in protecting against audiogenic seizures (clonic phase). Filled symbols indicate amino acid concentrations significantly different from control group. There is a very good correlation between the anticonvulsant efficacy (EDso against the clonic phase of audiogenic seizures) of a compound, and the ability of this compound to reduce cerebral aspartate levels in DBA/2 mice. A similar trend exists for the effect of these anticonvulsant valproate analogues on the cerebral GABA level, although the correlation is not as good between the anticonvulsant efficacy of these compounds and their ability to elevate forebrain GABA levels. Discussion Some straight-chain and branched chain fatty acids produce sedation and coma when injected i.v. into rats or rabbits (21,22), with potency increasing with chain length (C5-CI0). In the present study, this depression of activity
Vol.
32, No. 17, 1983
Valproate Analogues, Brain GABA and ASP
2029
was manifested as a dose-dependent reduction in the body temperature after
the i.p. administration of butyric, pentanoic, or hexanoic acid in DBAl2 mice, but was not accompanied by anticonvulsant activity in agreement with an earlier report that short, straight-chain fatty acids lack anticonvulsant activity against pentylenetetrazol seizures (23). Previous studies have shown that a number of short branched-chain fatty acid analogues of valproic acid exhibit anticonvulsant activity (24). All the known valproate metabolites (unsaturated or substituted valproate analogues) protect against electroshock or pentylenetetrazol seizures in mice, although with less potency (lo-90%) than valproate (25). Two M-substituted short fatty acids, 2-methyl-2-ethyl-caproic
) CHCOOH) may reflect on the mechanism of action of ~~YJr"ZYE.Z",i~?H'it sag & es ts a physico-chemical action on membranes rather than an action on a specific receptor/recognition However, such a physico-chemical action enzyme. the affinity or efficiency of a receptor/ionophore of an enzyme system that is membrane bound. Despite the acids their
similar anticonvulsant behavioural effects
action of these differ considerably.
site or active site of could secondarily modify complex or the activity
branched-chain Maitre et
fatty al
(26) have reported that the administration of an anticonvulsant dose of 2-methyl2-ethyl-caproic acid leads to loss of consciousness in mice, while an anticonvulsant dose of 2-2-dimethyl valeric acid has no behavioural effect. The present results show that among the four valproate analogues with the most potent anticonvulsant activity, two analogues (2-butyl-hexanoic acid and 2-propyl-heptanoic acid) produce comatose behaviour in the mice, while the other two (2-ethyl-hexanoic acid and 2-propyl-hexanoic acid) only produce
a slight
or
moderate
sedation
at
an anticonvulsant
dose
(2 moles/kg).
It is known that valproate administration elevates brain GABA levels and that although it remains to be valproate inhibits the GABA metabolizing enzymes, established how these effects relate to the anticonvulsant action of valMaitre et al (26) have shown that the administration of. proate (see 10).
2030
Valproate Analogues, Brain GABA and ASP
Vol. 32, No.
17, 1983
two anticonvulsant valproate analogues, 2-methyl-2-ethyl~caproic acid and 2-2-dimethyl valeric acid, leads to approximately 50% increases in forebrain GABA levels in mice and that these two analogues are more potent competitive inhibitors of GABA transaminase than valproate. However, the three saturated straight-chain fatty acids, propionic, butyric, and pentanoic acids, which lack anticonvulsant properties, are also more potent inhibitors of GABA transaminase than is valproic acid (28). Administration of some, but not all, of the anticonvulsant valproate metabolites leads to 5-25% increases in mouse forebrain GABA levels. There is no correlation between the anticonvulsant,,potency of the metabolites and the resulting GABA elevation, although Loscher et al (29) have reported a correlation between the anticonvulsant potency of these metabolites and the resulting increase in synaptosomal GABA content. Most of the anticonvulsant valproate analogues used in the present study produce 30-100% increases in mouse forebrain GABA levels. However, administration of two of the moderately potent anticonvulsant analogues, 2-methyl-pentanoic acid and 2-ethyl-butyric acid, is not associated with any increase in brain GABA level. Conversely, pentanoic acid administration leads to a 30% elevation in brain GABA level, although this straight chain analogue is lacking any significant anticonvulsant activity. There is a much stronger correlation between the anticonvulsant potency of these branched-chain fatty acids and their ability to reduce cerebral aspartate levels. The time-course for valproate-induced reduction in cerebral aspartate level is known to coincide with the period of protection against audiogenic seizures in mice (13). Following the administration of valproate to rats, there are 13-30% decreases in aspartate levels in all regions examined, in contrast to the GABA increase which was observed only in the cortex (9). The functional significance of the effect of valproate on cerebral aspartate metabolism is not known. However, in light of the close correlation demonstrated in the present study between the anticonvulsant potency of ten valproate analogues and their ability to depress brain aspartate levels, as well as the recent demonstration that antagonists of excitation induced by aspartate possess anticonvulsant properties (14,15), further studies in this area are likely to contribute to the understanding of the mechanism of action of valproic acid. Acknowledgements We thank the Medical Research Council of the United Kingdom and the Wellcome Trust for financial support. References I. 2. 3. 4. 5.
6. 7.
D. SIMON and J.K. PENRY, Epilepsia 16 549-573 (1975). P.M. JEAVONS, J.E. CLARK and M.C. MAHESHWARI, Dev. Med. Child. Neurol. 19 9-25 (1977). B.S. MELDRUM, Brain Res. Bull. 5 Suppl. 2, 579-584 (1980). R.L. MACDONALD and J.L. BARKER, Brain Res. 167 323-336 (1979). W. HAEFELY, L. PIERI, P. POLC and R. SCHAFFNER, Handbook of Experimental Pharmacology, 55/II, Eds. F. Hoffmeister and G. Stille, pp 13-262, Springer Verlag, Berlin (1981). B.S. MELDRUM, Psychopharmacology of Anticonvulsant Drugs, Ed. M. Sandler pp 62-78, Oxford University Press (1982). S. SIMLER, L. CIESIELSKI, M. MAITRE, H. RANDRIIANARISOA and P. MANDEL, Biochem. Pharmacol. 22 1701-1708 (1973).
Vol. 32, No. 17, 1983
8. 9. 10. 11.
12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22. 24.
25. 26. 27. 28. 29.
Valproate Analogues, Brain GABA and ASP
2031
G. ANLEZARK, R.W. HORTON, B.S. MELDRUM and M.C.B. SAWAYA, Biochem. Pharmacol. 25 413-417 (1976). A.G. CHAPMAN, K. RILEY, M.C. EVANS and B.S. MELDRUM, Neurochem. Res. 7 1089-1105 (1982). A.G. CHAPMAN, Progress in Epilepsy, Ed. F.C. Rose, Pitman Press, London, pp 371-383 (1983). P. MANDEL, S. SIMLER and L. CIESIELSKI, Psychopharmacology of Anticonvulsants, Ed. M. Sandler, pp 1.15, Oxford University Press, Oxford (1982). K. KUKINO and T. DEGUCHI, Chem. Pharm. Bull. 25 2257-2262 (1977). P.J. SCHECHTER, Y. TRANIER and J. GROVE, J. Neurochem. 31 1325-1327 (1978). M.J. CROUCHER, J.F. COLLINS and B.S. MELDRUM, Science 216 899-901 (1982). S.J. CZUCZWAR and B.S. MELDRUM, Europ. J. Pharmacol. 83 335-338 (1982). N. SEILER and S. SARHAN, Prog. Clin. Biol. Res. 39 425-439 (1980). R.L. COLLINS, Experimental Models of Epilepsy, Eds. D.P. Purpura, J.K. Penry, D. Tower, D.M. Woodbury and R. Walker, Raven Press, New York, pp 347-372 (1972). L. CIESIELSKI., M. MAITRE, C. CASH and P. MANDEL, Biochem. Pharmacol. 24 1055-1058. P.E. PFEFFER, L.S. SILBERT and J.M. CNIRINKO, J. Org. Chem. 37 451458 (1972). W.B. ESSMAN and F.N. SUDAK, Exp. Neurol. 9 228-235 (1964). F.E. SAMSON, N. DAHL and D.R. DAHL, J. Cl~n. Invest. 35 1291-1298 (1965) P.F. TEYCHENNE, I. WALTERS, L.E. CLAVERIA, D.B. CALNE, J. PRICE, B.B. McGILLIVARY and D. GOMPERTZ, Clin. Sci. Med. 50 463-472 (1976). H.J. KUPFERBERG, Antiepileptic Drugs: Mechanism of Action, Eds. G.H. Glaser, J.K. Penry, D.M. Woodbury, pp 643-654, Raven Press, New ~ork (1980). W. LOSCHER, Arch. Int. Pharmacodyn. 249 158-163 (1981). M. MAITRE, L. CIESIELSKI and P. MANDEL, Biochem. Pharmacol. 23 23632368 (1974). A. LESPAGNOL, T. MERCIER, F. ERB-DEBRUYNE and S. DESOIGEN, Annales Pharmaceutiques Francaises 30 193-198 (1972). L.J. FOWLER, J. BECKFORD and R.A. JOHN, Biochem. Pharmacol. 24 12671270,(1975). W. LOSCHER, G. BOHMME, H. SCHAFER and W. KOCHEN, Neuropharmacology 20 1187-1192 (1981).
Pergamon Press
ACUTE ANTICONVULSANT ACTIVITY OF STRUCTURAL ANALOGUES OF VALPROIC ACID AND CHANGES IN BRAIN GABA AND ASPARTATE CONTENT Astrid G. Chapman 1, B r i a n S. Meldrum 2 and E t i e n n e Mendes 3 Department of Neurology I, Rayne Institute, King's College Hospital Medical School, 123 Coldharbour ~ane, London, SE5 9NU. Department of Neurology-, Institute of Psychiatry, De Crespigny Park, London, ~E5 8AF, U.K. Sanofi Recherche , 195 Route d'Espagne, 31300 Toulouse Cedex, France. (Received in final form January 31, 1983) Summary Ten analogues of valproic acid (substituted butyric, pentanoic and hexanoic acids) were tested for anticonvulsant activity against audiogenic seizures in DBA/2 mice. There is a consistent correlation between the structure of these branched-chain fatty acids and their anticonvulsant potency, the larger molecules being the m o ~ a c t i v e . There is also a strong correlation between the anticonvulsant potency of these compounds and their ability to reduce cerebral aspartate levels. Cerebral GABA levels are elevated by most, but not all, of the actively anticonvulsant valproate analogues. The anticonvulsant activity of valproic acid (2-propyl-pentanoic acid) or its sodium or magnesium salts, has been known since 1963. Valproate is currently used to treat several syndromes of epilepsy (see I-2) but its mechanisms of action remains essentially unknown (3). The principal inhibitory neurotransmitter in the mammalian brain is GABA (4-aminobutyric acid). Benzodiazepines and barbiturates are thought to owe at least part of their anticonvulsant action to enhancement of the postsynapic inhibitory action of GABA (4-6). Brain GABA content is increased after the acute administration of valproate to rodents (7-9). In vitro valproate inhibits all of the three enzymes involved in the further metabolism of GABA (GABAtransaminase, succinic semialdehyde dehydrogenase, and aldehyde reductase) but its in vivo effect on GABA metabolism is uncertain (see 10). Nevertheless the biochemical data have been interpreted in terms of valproate enhancing GABAergic inhibition by making more transmitter available for synaptic release (11). The acute administration of valproate also decreases brain aspartate content (9,12-13). The possible significance of this effect has been recently emphasized by the discovery of the anticonvulsant properties of amino acid analogues that preferentially block excitation due to N-methyl-D-aspartate or aspartic acid Cas opposed to excitation due to kainic acid, quisqualic acid or glutamate) (14-15). The effects of valproate on GABA and aspartate appear to be independent. Changes in concentration of GABA and aspartate 0024-3205/83/172023-09503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
2024
Valproate
Analogues,
Brain G A B A and ASP
Vol.
32, No.
17, 1983
show a different gross regional distribution in the rat brain (9) and a different s u b c e l l u l a r d i s t r i b u t i o n in brain homogenates (16). We have examined the acute a n t i c o n v u l s a n t action, of a series of valproate analogues (i.e. b r a n c h e d - c h a i n fatty acids with carbon chain lengths of 4-7) in DBA/2 mice showing s o u n d - i n d u c e d seizures that are age dependent (17). For comparative purposes we have also included 3 straight chain fatty acids. A time point of 30 min was selected for drug evaluation as the effect of valproic acid against a u d i o g e n i c seizures is fully established at this time (13, 18), and the sedative and other side effects of the s t r a i g h t - c h a i n and branchedchain analogues show a similar time course to those of valproic acid. We have also measured forebrain aspartate and GABA concentrations after drug treatments to assess the correlation of such changes with a n t i c o n v u l s a n t action. Methods All experiments were performed Psychiatry from LAC stock.
in DBA/2 mice
bred at the Institute
of
For e v a l u a t i o n of the behavioural and a n t i c o n v u l s a n t drug a c t i o n mice of either sex, 21-26 days old (body weights 6-13 g) were injected with olive oil (B.P. Boots Ltd.) 0.1 ml, or with the test compound, 0.5-4 mmoles/kg, dissolved in 0.1 ml olive oil. Thirty min after drug a d m i n i s t r a t i o n rectal temperature was measured and the animal placed under a perspex dome (diameter 58 cm) for observation of motor activity. Stimulation by an electric bell (Friedland chimes, 3 inch diameter, p r o d u c i n g 109 dB at mouse level) was applied for 60 sec or until tonic extension occurred. Time to onset of each phase of the seizure response was recorded. A seizure response (SR) score was calculated for each animal on the basis of the maximal response:0 = none; I = wild running; 2 = clonus; 3 = tonic extension; 4 = respiratory arrest. Later, sequential, phases are indicated by higher scores. Later phases were never seen in the absence of the p r e c e d i n g (lower score) phase. For groups of 8-10 mice receiving one dose of drug, differences in the occurrence of seizure phases (compared with carrier treated litter mate mice, run at the same time) were evaluated with Fisher's exact test. For each phase of the seizure response, the EDeN for s u p p r e s s i o n of that phase was calculated g r a p h i c a l l y (using 4 l o g a r i t ~ i c a l l y augmented doses). The drug dose reducing the SR score by 50% was also calculated. For biochemical studies mice were injected i n t r a p e r i t o n e a l l y with 2 m m o l e s / k g of each acid in olive oil, 0.1 ml, 30 min prior to d e c a p i t a t i o n into liquid nitrogen. The heads were kept in liquid nitrogen until the forebrains were chisled out on the same day with liquid N_ irrigation and extracted with 5 volumes of 0.4 N HCI04, I mM EDTA and s u b s e q u e n t l y neutralized with a KOHNaHCO 3 mixture. Aspartate levels in the extract were determined e n z y m a t i c a l l y using the malate dehydrogenase (MDH) linked assay. The concentrations in the assay mix (I ml) were: Tris. HC1, pH 8.0:60 mM, ~ - k e t o g l u t a r a t e : O . 2 mM, NADH:O.I mM, MDH:2.5 ~ g / m l . Thirty ~l brain extract or standard aspartate solution (1050 nmoles) was used per ml of assay mix. The reactions were started with the addition of g l u t a m a t e - o x a l o a c e t a t e amino-transferase (GOT) and the decrease in absorbance at 340 mm was determined s p e c t r o p h o t o m e t r i c a l l y . GABA levels were determined f l u o r o m e t r i c a l l y using the GABAase assay. The concentrations in the final reaction mix (2.5 ml) were: Na pyrophosphate buffer, pH 8.6: 60 mM, NADP+:IO mM, m e r c a p t o e t h a n o l : 6 mM, ~ - k e t o g l u t a r a t e : 2 mM. The reaction mixture contained 0.25 ml brain extract or standard GABA (20-150 nmoles/ 2.5 ml) solution, and the reaction was initiated by the addition of
Vol. 32, No. 17, 1983
Valproate Analogues,
Brain GABA and ASP
2025
-ketoglutarateo The analogues listed in Table I were either obtained from commercial sources and repurified by distillation, or synthesised in the laboratories of Sanofi Recherche, Toulouse, by malonic synthesis (for methyl-2-pentanoic acid) or ~-alkylation of the corresponding carboxylic acid as described by Pfeffer et al (19). The purity of all the analogues was determined by gas chromatography, infrared and NMR characteristics, and by elementary analysis. Results A typical audiogenic seizure in DBA/2 mice consists of a wild running phase (latency I-6 sec), a clonic phase (latency 5-15 sec), and tonic extension (latency 10-35 sec), frequently followed by respiratory arrest (latency 25-50 sec). Figure I shows the percentage of DBA/2 mice (n = 8-10 per dose) undergoing the sound-induced wild running and clonic phases following the intraperitoneal administration of a series of valproate analogues (0.5-4 mmoles/kg; 30 min). Similar response curves, indicating slightly lower effective doses of the analogues, were obtained for the tonic extension and respiratory arrest phases of the seizure (data not shown). The anticonvulsant effect of valproic acid is shown by curve 7 in Figure I. Four of the valproate analogues, 2-ethyl-hexanoic acid, 2-propyl-hexanoic acid, 2-propylheptano~c acid, and 2-butyl-hexanoic acid (curves 8-11) are more potent anticonvulsants than valproate. The short, straight-chain fatty acids, butyric, pentanoic, and hexanoic acid, as well as 2-ethyl-pentanoic acid (curves I-4) have little or no anticonvulsant properties. 2-Methyl-pentanoic acid and 2-ethyl-butyric acid display intermediate anticonvulsant activity (curves 5-6).
WILD
RUNNING
CLONIC PHASE
!
1,2,3
100
80
7
1
s
80
uJ
u) 60
60
Z O
o_ 40
6
4(]
IJJ
Oc
20
20
o
0
!
!
!
!
|
05
I
2
4
0.5
mmol/kg
|
!
I
1
2
4
mmol / kg
FIG. I Effect of valproate analogues (0.5 - 4 mmoles/kg, i.p. 30 min prior to test) on the initial phases of the sound induced seizure response. Fatty acids indicated are: I. hexanoic acid; 2. pentanoic acid; 3. 2-ethyl-pentanoic acid; 4. butyric acid; 5. 2-methyl-pentanoic acid; 6. 2-ethyl-butyric acid; 7. ~alproic acid; 8. 2-ethyl-hexanoic acid; 9. 2-propyl-hexanoic acid; 10. 2-propylheptanoic acid; 11. 2-butyl-hexanoic acid.
2026
Valproate
Analogues,
Brain GABA and ASP
Vol.
32, No.
17,
1983
The inactive or w e a k l y a n t i c o n v u l s a n t compounds (I-6 in Fig. I) have little or no effect on the behaviour of the mice in doses up to 2 mmoles/kg. At mmoles/kg these compounds produce a slight sedation, piloerection, and peritoneal irritation, as well as a slight ata×ia in some cases. Valproic acid produces no behavioural effects at doses of 0.5 and I mmole/kg, a slight ataxia at 2 mmcles/kg, and heavy sedation and ataxia at 4 mmoles/kg. The four potent a n t i c o n v u l s a n t analogues (8-11 in Fig. I) produce no behavioural effects at 0.5 mmoles/kg, and slight sedation with fairly normal exploration at I mmole/kg. At 2 mmole/kg 2 - e t h y l - h e x a n o i c acid produces slight sedation and no ata×ia, whereas 2 - p r o p y l - h e x a n o i c acid produces moderate sedation and slight ataxia. The mice receiving 2 m m o l e s / k g 2 - b u t y l - h e × a n o i c acid or 2p r o p y l - h e p t a n o i c acid were comatose, and 2 mice died in the latter group. I00
so
R/CHCOOH
I
LLI 1 0 0 U') < -r"
o
o
[1_ 0
~_
50
HCOOH
0 _.J 0
7 0
--
0._ 100 O3 LLI
o
o
L
5o
I
R:
ox
H
|
I
f
q%
R"
HCOOH
|
gH,,
FIG. 2 Relationship between chain length and a n t i c o n v u l s a n t potency of valproate analogues. The effect of I m m o l e / k g of valproate analogues (i.p. 30 min) on the clonic phase of audiogenic seizures in DBA/2 mice. The filled symbol denotes valproate. R = Alpha s u b s t i t u t i o n for three formulae illustrated.
Vol. 32, No. 17, 1983
Valproate
Analogues, Brain GABA and ASP
2027
The administration of valproate and all the valproate analogues resulted in a dose-dependent reduction in the rectal temperature of the mice: Control value: 37.2~0.06 °, 0.5 mmoles/kg: 36.930.1 ° , I mmole/kg: 36.430.1 °, 2 mmoles/ kg: 35.3~O.6 °, and 4 mmoles/kg: 34.7~O.2 °. The reductions were similar for the active and the inactive analogues, and they were not severe enough to affect the audiogenic seizure response (20). Figure 2 shows the relationship between the structure of the valproate analogues and their anticonvulsant potency expressed as clonic seizure response observed following the administration of I mmole/kg of these analogues. The anticonvulsant potency of these branched-chain fatty acids is proportional to the chain length of the side group for all the three branched fatty acid series: substituted hexanoic, pentanoic, and butyric acid. The anticonvulsant potencies of the valproate analogues, expressed as ED (mM) against the clonic phase of the audiogenic seizures and against the 50 overall seizure response (SR) score are listed in Table I, along with the forebrain levels of GABA and aspartate determined 30 min after the administration of 2 mmoles/kg of these compounds. TABLE I (A) ED50 for Suppression of Audiogenic Seizures in DBA/2 Mice by Valproate Analogues and (B) the Effect of Administration of these Analogues (2 mmoles/kg i.p., 30 min) on the Aspartate and GABA Levels in Forebrain.
(A) Fatty Acid
Anticonv. WR
Control Hexanoic Pentanoic Butyric 2-Ethyl-Pentanoic 2-Methyl-Pentanoic 2-Ethyl-Butyric Valproic 2-Propyl-Heptanoic 2-Propyl-Hexanoic 2-Butyl-Hexanoic 2-Ethyl-Hexanoic
7.0 8.0 4.0 2.8 1.4 0.72 0.85 0.75 0.90
Potency
(B) (ED5omM)
Clonic
SR
10.0 5.8 5.6 4.8 4.0 2.6 1.25 0.72 0.68 0.68 0.66
8.0 6.0 5.0 5.0 2.7 2.4 1.1 0.7 0.7 0.7 0.5
Brain Conc. Aspartate 2.7730.13 2.86-0.14 2.2330.18 * 3.0630.13 2.2130.15" 2.3030.11" 2.1030.11" 1.3030.12"** 1.2530.06 *** 1.3230.08"** 1.2730.06 *** 0.9830.11"**
(~moles/g) GABA 1.4530.06 1.4030.04 2.06-0.14" 1.5330.08 2.0930.05 *** 1.5730.08 1.5830.10 2.7730.20 *** 2.0630.09*** 1.8330.08 ** 2.1930.05 *** 2.9430.14"**
Valproate analogues are listed in order of increasing + overall anticonvulsant efficacy. Amino acid values are expressed as mean - SEM; statistical difference from control values (Student's t-test): *p < 0.05; ** p < 0.01; *** p < 0.001. SR : Seizure response score (see methods). The administration of valproic acid and the four most potent anticonvulsant analogues, 2-propyl-heptanoic acid, 2-propyl-hexanoic acid, 2-butyl-hexanoic acid, and 2-ethyl-hexanoic acid results in 52-65% reductions in the aspartate levels and 26-103% increases in the GABA levels in the forebrain.
2028
Valproate Analogues,
Brain GABA and ASP
Vol. 32, No. 17, 1983
The relationship between the anticonvulsant potency of the valproate analogues and their effect on the forebrain amino acid levels is illustrated graphically in Figure 3.
{ 0
E I < n7
<
o ,
,
,
,
,
,
,
,
,
,
,
'
lb
, , f /
,
{
i 0
E-j 0_ U~
<_
6
'
'
'
¢
'
;s
CdNrROL
EDso,mM. CLONIC PHASE FIG. 3 Cerebral aspartate and GABA levels following the administration of valproate analogues (2 mmoles/kg i.p., 30 min) to DBA/2 mice, versus the potency of these analogues in protecting against audiogenic seizures (clonic phase). Filled symbols indicate amino acid concentrations significantly different from control group. There is a very good correlation between the anticonvulsant efficacy (EDso against the clonic phase of audiogenic seizures) of a compound, and the ability of this compound to reduce cerebral aspartate levels in DBA/2 mice. A similar trend exists for the effect of these anticonvulsant valproate analogues on the cerebral GABA level, although the correlation is not as good between the anticonvulsant efficacy of these compounds and their ability to elevate forebrain GABA levels. Discussion Some straight-chain and branched chain fatty acids produce sedation and coma when injected i.v. into rats or rabbits (21,22), with potency increasing with chain length (C5-CI0). In the present study, this depression of activity
Vol.
32, No. 17, 1983
Valproate Analogues, Brain GABA and ASP
2029
was manifested as a dose-dependent reduction in the body temperature after
the i.p. administration of butyric, pentanoic, or hexanoic acid in DBAl2 mice, but was not accompanied by anticonvulsant activity in agreement with an earlier report that short, straight-chain fatty acids lack anticonvulsant activity against pentylenetetrazol seizures (23). Previous studies have shown that a number of short branched-chain fatty acid analogues of valproic acid exhibit anticonvulsant activity (24). All the known valproate metabolites (unsaturated or substituted valproate analogues) protect against electroshock or pentylenetetrazol seizures in mice, although with less potency (lo-90%) than valproate (25). Two M-substituted short fatty acids, 2-methyl-2-ethyl-caproic
) CHCOOH) may reflect on the mechanism of action of ~~YJr"ZYE.Z",i~?H'it sag & es ts a physico-chemical action on membranes rather than an action on a specific receptor/recognition However, such a physico-chemical action enzyme. the affinity or efficiency of a receptor/ionophore of an enzyme system that is membrane bound. Despite the acids their
similar anticonvulsant behavioural effects
action of these differ considerably.
site or active site of could secondarily modify complex or the activity
branched-chain Maitre et
fatty al
(26) have reported that the administration of an anticonvulsant dose of 2-methyl2-ethyl-caproic acid leads to loss of consciousness in mice, while an anticonvulsant dose of 2-2-dimethyl valeric acid has no behavioural effect. The present results show that among the four valproate analogues with the most potent anticonvulsant activity, two analogues (2-butyl-hexanoic acid and 2-propyl-heptanoic acid) produce comatose behaviour in the mice, while the other two (2-ethyl-hexanoic acid and 2-propyl-hexanoic acid) only produce
a slight
or
moderate
sedation
at
an anticonvulsant
dose
(2 moles/kg).
It is known that valproate administration elevates brain GABA levels and that although it remains to be valproate inhibits the GABA metabolizing enzymes, established how these effects relate to the anticonvulsant action of valMaitre et al (26) have shown that the administration of. proate (see 10).
2030
Valproate Analogues, Brain GABA and ASP
Vol. 32, No.
17, 1983
two anticonvulsant valproate analogues, 2-methyl-2-ethyl~caproic acid and 2-2-dimethyl valeric acid, leads to approximately 50% increases in forebrain GABA levels in mice and that these two analogues are more potent competitive inhibitors of GABA transaminase than valproate. However, the three saturated straight-chain fatty acids, propionic, butyric, and pentanoic acids, which lack anticonvulsant properties, are also more potent inhibitors of GABA transaminase than is valproic acid (28). Administration of some, but not all, of the anticonvulsant valproate metabolites leads to 5-25% increases in mouse forebrain GABA levels. There is no correlation between the anticonvulsant,,potency of the metabolites and the resulting GABA elevation, although Loscher et al (29) have reported a correlation between the anticonvulsant potency of these metabolites and the resulting increase in synaptosomal GABA content. Most of the anticonvulsant valproate analogues used in the present study produce 30-100% increases in mouse forebrain GABA levels. However, administration of two of the moderately potent anticonvulsant analogues, 2-methyl-pentanoic acid and 2-ethyl-butyric acid, is not associated with any increase in brain GABA level. Conversely, pentanoic acid administration leads to a 30% elevation in brain GABA level, although this straight chain analogue is lacking any significant anticonvulsant activity. There is a much stronger correlation between the anticonvulsant potency of these branched-chain fatty acids and their ability to reduce cerebral aspartate levels. The time-course for valproate-induced reduction in cerebral aspartate level is known to coincide with the period of protection against audiogenic seizures in mice (13). Following the administration of valproate to rats, there are 13-30% decreases in aspartate levels in all regions examined, in contrast to the GABA increase which was observed only in the cortex (9). The functional significance of the effect of valproate on cerebral aspartate metabolism is not known. However, in light of the close correlation demonstrated in the present study between the anticonvulsant potency of ten valproate analogues and their ability to depress brain aspartate levels, as well as the recent demonstration that antagonists of excitation induced by aspartate possess anticonvulsant properties (14,15), further studies in this area are likely to contribute to the understanding of the mechanism of action of valproic acid. Acknowledgements We thank the Medical Research Council of the United Kingdom and the Wellcome Trust for financial support. References I. 2. 3. 4. 5.
6. 7.
D. SIMON and J.K. PENRY, Epilepsia 16 549-573 (1975). P.M. JEAVONS, J.E. CLARK and M.C. MAHESHWARI, Dev. Med. Child. Neurol. 19 9-25 (1977). B.S. MELDRUM, Brain Res. Bull. 5 Suppl. 2, 579-584 (1980). R.L. MACDONALD and J.L. BARKER, Brain Res. 167 323-336 (1979). W. HAEFELY, L. PIERI, P. POLC and R. SCHAFFNER, Handbook of Experimental Pharmacology, 55/II, Eds. F. Hoffmeister and G. Stille, pp 13-262, Springer Verlag, Berlin (1981). B.S. MELDRUM, Psychopharmacology of Anticonvulsant Drugs, Ed. M. Sandler pp 62-78, Oxford University Press (1982). S. SIMLER, L. CIESIELSKI, M. MAITRE, H. RANDRIIANARISOA and P. MANDEL, Biochem. Pharmacol. 22 1701-1708 (1973).
Vol. 32, No. 17, 1983
8. 9. 10. 11.
12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22. 24.
25. 26. 27. 28. 29.
Valproate Analogues, Brain GABA and ASP
2031
G. ANLEZARK, R.W. HORTON, B.S. MELDRUM and M.C.B. SAWAYA, Biochem. Pharmacol. 25 413-417 (1976). A.G. CHAPMAN, K. RILEY, M.C. EVANS and B.S. MELDRUM, Neurochem. Res. 7 1089-1105 (1982). A.G. CHAPMAN, Progress in Epilepsy, Ed. F.C. Rose, Pitman Press, London, pp 371-383 (1983). P. MANDEL, S. SIMLER and L. CIESIELSKI, Psychopharmacology of Anticonvulsants, Ed. M. Sandler, pp 1.15, Oxford University Press, Oxford (1982). K. KUKINO and T. DEGUCHI, Chem. Pharm. Bull. 25 2257-2262 (1977). P.J. SCHECHTER, Y. TRANIER and J. GROVE, J. Neurochem. 31 1325-1327 (1978). M.J. CROUCHER, J.F. COLLINS and B.S. MELDRUM, Science 216 899-901 (1982). S.J. CZUCZWAR and B.S. MELDRUM, Europ. J. Pharmacol. 83 335-338 (1982). N. SEILER and S. SARHAN, Prog. Clin. Biol. Res. 39 425-439 (1980). R.L. COLLINS, Experimental Models of Epilepsy, Eds. D.P. Purpura, J.K. Penry, D. Tower, D.M. Woodbury and R. Walker, Raven Press, New York, pp 347-372 (1972). L. CIESIELSKI., M. MAITRE, C. CASH and P. MANDEL, Biochem. Pharmacol. 24 1055-1058. P.E. PFEFFER, L.S. SILBERT and J.M. CNIRINKO, J. Org. Chem. 37 451458 (1972). W.B. ESSMAN and F.N. SUDAK, Exp. Neurol. 9 228-235 (1964). F.E. SAMSON, N. DAHL and D.R. DAHL, J. Cl~n. Invest. 35 1291-1298 (1965) P.F. TEYCHENNE, I. WALTERS, L.E. CLAVERIA, D.B. CALNE, J. PRICE, B.B. McGILLIVARY and D. GOMPERTZ, Clin. Sci. Med. 50 463-472 (1976). H.J. KUPFERBERG, Antiepileptic Drugs: Mechanism of Action, Eds. G.H. Glaser, J.K. Penry, D.M. Woodbury, pp 643-654, Raven Press, New ~ork (1980). W. LOSCHER, Arch. Int. Pharmacodyn. 249 158-163 (1981). M. MAITRE, L. CIESIELSKI and P. MANDEL, Biochem. Pharmacol. 23 23632368 (1974). A. LESPAGNOL, T. MERCIER, F. ERB-DEBRUYNE and S. DESOIGEN, Annales Pharmaceutiques Francaises 30 193-198 (1972). L.J. FOWLER, J. BECKFORD and R.A. JOHN, Biochem. Pharmacol. 24 12671270,(1975). W. LOSCHER, G. BOHMME, H. SCHAFER and W. KOCHEN, Neuropharmacology 20 1187-1192 (1981).