TZPS - April 1988 [Vol. 91
`
`including
`ethosux-
`
`imide and trimethadione”. These epileptic phenomena may be con- sidered as
`
`the result of a hyper-
`endogenous brain
`account
`
`its pharmacological and biochem- ical properties, we would suggest that GHB plays a role in the resultant hyperactivity. Thus, we propose that the mechanism of action of some antiepileptic drugs is due to their interactions with the GHB system in the brain. We shall comer&ate our attention on the specific effects of valproate on cerebral GHB functions
`propose that these biochemical actions indicate a heuristic mol- ecular model for the mode of action of certain anticonvulsant drugs. Anticonvulsant
`
`mechanism of
`valproate: hypothesis
`
`and
`
`rat brain hippocampus4. More- over, its half-life in rat brain (about 28 min) is at least as short as those of established neuro- transmitters. Administration of GHB to ani- mals and humans induces various neuropharmacological and neuro- physiological effects, the most salient of which are: (1) modula- tion of dopaminergic activity’ (especially in the striatum) and an increase in 5-HT tumovefi, and (2) a pronounced induction of seda- tion leading at higher GHB doses to loss of consciousness and anaesthesia’. This latter property has been exploited clinically. However, our principal interest here lies in the electroencephalo- graphic perturbations induced by GHB administration. These effects have been most studied in the cat* and in the rat’. In these animals, GHB induces authentic experi- mental epilepsies which are char- acterized by mono- and poly- phasic discharges. These dis- charges are analogous to those observed in human petit ma1 epilepsy lo. The abnormal elec- troencephalograms induced by GHB are antagonized by various 127 anticonvulsant drugs
`effect against various forms of experimental epilepsy. This GABA increase has been ascribed to the inhibitory effect of valpro- ate on the GABA shunt enzymes, GABA transaminase” and succi- nit semialdehyde dehydrogenase13 (see Fig. 1). The inhibitory effect is more pronounced on the latter enzyme and is competitive with respect to the substrate, succinic semialdehyde13 - a metabolite which has only been detected in minute quantities in brain tissue. Valproate has no effect on GABA release from cortical synapto- somes preloaded with labelled GABA14. Concomitant with the GABA level increase induced by val- proate is a marked reduction in cerebral aspartate level”. However, no interaction of val- proate with possible excitatory synapses which function with aspartate has been demonstrated. The increase in the cerebellar cGMP level frequently observed in numerous forms of experimen- tal epilepsy is greatly diminished by prior administration of valpro- ate, whereas CAMP levels are unchanged16. This increase in cGMP level is considered as play- ing a role in the induction and continuation activity”. of epileptogenic
`
`Is the anticonvulsant mechanism of valproate linked to its interaction with the cerebral y-hydroxybutyrate system? Philippe Vayer, Christopher D. Cash and Michel Jvlaitre
`
`(GHB) may be a neuro-
`that y-hydroxybutyrate
`evidence
`is
`There
`modulator in the CNS. Administration of this compound to various mammals
`at sub-anaesthetic doses induces brain electrical activity resembling
`human absence epilepsy. This effect
`is antagonized by the anticonvulsant
`drugs valproate and ethosuximide, and by the opiate antagonist naloxone.
`valproate and ethosuximide reduce the depolarization-induced
`release of
`In vitro
`GHB from rat hippocampal
`slices, and
`valproate antagonizes
`the
`in hippocampal cGMP levels induced by prior GHB administration.
`therefore propose
`that the anticonvulsant
`action of valproate may be linked to its interaction with the endogenous
`system.
`
`In rodents, valproate induces an increase in the cerebral GABA 1eve112, coinciding with a protec-
`
`rivrt
`
`y-Hydroxybutyrate (GHB) is a iiermal brain metabolite derived primarily from GABA. Recent bio- chemical and pharmacological data (see Ref. 1) lend support to the hypothesis that this substance may play a neuromodulator role in brain. GHB is heterogenously distri- buted in brain tissue, where it is synthesized by a specific enzyme located exclusively in neurones. The highest concentration of this substance is found in the synapto- somal fraction2. GHB is released by depolarization of brain tissue slices by a Ca2+-dependent mechanism and is transported by a high-affinity, energy- and Na+- dependent system. High-affinity specific GHB binding sites are primarily located on the rostra1 area of the brain, the richest region being the hippocampus3. In vivo, GHB administration induces accumulation of cGMP in
`
`and Christopher Cash are
`Philippe Vayer
`postdoctoral research scientists (it the Centre
`de Neurochimie du CNRS and Unit6 INSERM
`U44. 5 rue Blaise Pascal, 67084 Strasbourg,
`France. Michel Maitre
`Is an Associate Profes-
`sor at Me Faculty of Medicine, Uniue~aitB
`Louis hsteur,
`67084 Strarbourg, France,
`
`Finally,
`valproate has no effect @ lYgU, L’lscvlsr Publlcatluns, Cambtidgc Ill65 - 6147/88/$02.01)
`
`PAR1012
`IPR of U.S. Patent No. 8,772,306
`Page 1 of 3
`
`now
`that of
`in vivo
`increase
`Michel Maitre and colleagues
`GHB
`valproate and also by
`activity of an
`system and, taking into
`

`
`128
`
`GABA
`
`CABA-T ’ k Fig.
`
`1. The
`GABA shwnnt and
`Krebs cyc/e in the
`mitochondnon. Valpro-
`(represinted
`by
`aclssors) blocks the oxidation
`swccihic semialdehyde (SSA)
`by swccihic semialdehyde
`to succihate
`The increased
`dehydrogenase
`of succinic semialdehyde may either
`or be Wansaminaled to GABA, leading in both
`cases lo raised GABA levels.
`
`inhibit GABA-Wansaminare
`
`TIPS - April 1988 [Vol. 91
`
`into the
`
`out of the mitochondria
`cytoplasm (Fig. 1).
`A more plausible mechanism for
`valproate-induced
`increases
`in
`GHB levels is the powerful inhibi-
`tion of nonspecific succinic semi-
`aldehyde reductase by valproate2’.
`[This enzyme is identical to the
`previously described ALR1, also
`referred to as glucuronate reduc-
`tase or SSRl (Ref. 22).] It is reported
`to be responsible for the catabol-
`ism of GHB to succinic semialde-
`hyde both in vitro22 and in vivo23.
`Under physiological conditions in
`is degraded to GABA
`vitro,
`via nonspecific
`succinic
`semi-
`aldehyde
`reductase and GABA
`transaminase2’. The operation of
`this catabolic pathway explains
`why
`inhibitors of nonspecific
`semialdehyde reductase such as
`valproate or ethosuximide,
`or
`GABA
`transaminase
`inhibitors
`such as y-vinyl GABA, increase
`GHB levels2 (Fig 2).
`ethosuximide
`Valproate
`and
`also considerably reduce the Ca2+-
`dependent depolarization-induced
`release of GHB from hippocampal
`and striatal slices frcm rat brain24.
`The ICsos for these two drugs in
`this test are compatible with their
`brain levels after administration
`of anticonvulsant doses.
`The latter phenomenon explains
`why the epileptogenic effects of
`GHB administration are attenu-
`ated by valproate or ethosuxi-
`mide’l, and also explains the para-
`dox that drugs such as valproate
`increase GHB brain levels25 whilst
`antagonizing its effects.
`The increase in cGMP levels in
`certain brain regions (such as the
`hippocampus)
`commonly
`seen
`after administration of convulsant
`drugs
`is concomitant with
`the
`
`which reduces succinic semialde-
`hyde to GHB (specific succinic
`semialdehyde
`reductase)
`is un-
`affected by valproate2’. In fact the
`increase in GABA levels induced
`by valproate has been ascribed to
`succinic semialdeh de dehydro-
`inhibition
`genase
`However,
`succinic semialdehyde dehydro-
`genase is a mitochondrial enzyme
`whereas specific succinic semi-
`aldehyde reductase is cytoplasmic.
`Thus the hypothesis that valproate
`increases GHB levels via an in-
`creased synthesis due to higher
`precursor availability would de-
`pend on an as yet unknown
`mechanism whereby excess suc-
`cinic semialdehyde is transported
`
`on the GABA* receptor nor on
`benzodiazepine receptors, a&tough
`it displaces the convulsant dihydro-
`picrotoxin from its binding site”.
`In general, it is thought that
`valproate potentiates GABAergic
`neurotransmission. However,
`it
`decreases its turnover rate and it
`does not modify its release. A
`possible direct GABA-like agonist
`action of valproate on a post-
`synaptic
`site has yet
`to be
`demonstratedls, although a direct
`action of sodium valproate on
`action potentials of cultured neur-
`ons has been reported19.
`
`Interactions of valproate with
`cerebral GHB system
`Several workers have reported
`that valproate administration
`to
`rodents brings about an increase
`in the brain GHB level”. This
`increase
`is
`time-
`and dose-
`dependent, and appears to be due
`to
`two
`related
`factors: direct
`inhibition of catabolic enzymes
`and a
`reduction
`in
`synaptic
`release.
`As the principal precursor of
`GHB is GABA, the mechanism of
`this GHB increase could be due to
`inhibition of the mitochondrial
`enzyme succinic semialdehyde de-
`hydrogenase (EC1.2.1.24), which
`causes the pool of succinic semi-
`aldehyde (the direct precursor of
`GHB) to be elevated. The enzyme
`
`GlU i 2 BBA -----------cGHB % BSA valproste ethoauximide
`
`fig. 2. lnhlbition of GHB carabol~sm by
`redwctase; (2),
`(GVG).
`(l), Speclflc swccinic semlaldehyde
`semiardehyde redwctase; (3), GABA-Wansamlnase.
`direcfly inhibit nonspecific swccinic semieldehyde redwctase, whereas QVG
`inh/b/ts
`GABA-transaminase leading to an increase In swcdnic semialdehyde (SSA), which is a
`product khibkor of the dehydwgenase.
`
`and y-vinyl GABA
`
`PAR1012
`IPR of U.S. Patent No. 8,772,306
`Page 2 of 3
`
`c
`of
`(SSADH).
`pool
`(GABA-l)
`z
`.
`GHB
`valproate, ethosuxlmlde
`nonspeciflo swccinic
`Valproeta and ethoruxlmlde
`

`
`Ptvposad
`and
`
`chain-breaking
`
`is
`
`of
`action
`3.
`Fig.
`on the GM
`val~roate
`sysbm:
`inhibited,
`and
`GHB-
`endorphin
`of
`refaetse
`term-
`opioid-paptida
`prasynapfic
`activafed
`(NLX)
`is not stimulafsd. Naloxone
`inafs
`antagonizes Ihe action
`of peptidergic synap
`directly
`and
`thus
`indiractly
`antagonizes
`the epileptic
`phenomena
`induced
`by
`GHB.
`
`5 Roth. R. I-L
`
`605-610
`
`1.
`
`Eur.
`
`Biochem.
`
`Ann.
`
`Neural.
`
`6, 296-310 18 Chapman, A., Keane, P. E., Meldrum, B.S., Simiand, J. and Vemieres, J. C. (1982) Prog.
`
`19,315-359
`
`Neurobiol.
`
`April
`
`1988 EVol. 91 u -
`
`cGMP increase ses
`
`commencement and generaliza- tion of massive depolarization phenomena”. The increase we have observed in hippocampal cGMP after GHB administration* could be considered as either a side-effect or a precursor to the evolution of the epilepsy induced by GHB, the latter directly or indirectly provoking a perturba- tion in the membrane polarization of the hippocampus, a region rich in high-affinity GHB binding sites3. In various experimental epilepsies, therapeutic doses of vslproate antagonize increases in cGMP levels; valproate also antag- onizes, both
`
`in vivo
`
`Doherty, J. I’. and Wakers, J. R. (1980) Brain Res. 189,556-560 6 Hedner, Tb. and Lundborg, P. (1983) I. Neural Transm. 57,3= 7 Laborit, H. (1964) Int. J. Neurophnrma- co/. 3, 433-452 8 Winters. W. D. and Spooner, C. E. (1965) Int. 1. Neurophnrmucof. 4,197-200 9 Marcus, R. J., Winters, W. D., Mori, K. and Spooner, C. E. (1967) ht. 1. Pharma- col. 6, 175-185 10 Godschalk, M., Dzoljic, M. R. and Bonta, I. L. (1977)
`Pharmacol. 44, 105-111 11 Godschalk, M., Dzoljic, M. R. and Bonta, I. L. (1976) Neurosci. Left. 3, 145- 150 12 Simler, S., Ciesielski, L., Maitre, M., Randrianarosa, H. and Mandel, P. (1973) Biochem. Pharmacol. 22, 1701- 1708 13 Van der Laan, J. W. and De Boer, Th. (1979) 1. Neurochem. 32,1769-1780 14 AbduI-Ghani, A. S., Coutinho-Netto, J., Druce, D. and Bradford, H. F. (1981)
`Pharmacol. 30,363-368 15 Schechter, P. J., Tranier, Y. and Grove, J. (1978) J. Neurochem. 31,1325-1327 16 McCandless, D. W., Feussner, G. K., Lust, W. D. and Passoneau. J. V. (1979) J. Neurochem. 32,755-760 17 Gross, R. A. and Ferrendelli, J. A. (1979)
`
`48, 196-201 3 Hechler, V., Weiasman, D., Mach, E., Pujol, J. P. and Maitre, M. (1987) /, Neurochem. 49, 1025-1032 129 4 Vayer, I’., GobaiIIe, S., Mandel, P. and Maitre. M. (1987) Life Sci. 41,
`and in vitro, the increase in cGMP levels seen after GHB administration*. Etho- suximide also antagonizes this increase* and thus it can be infer- red that . their anticonvulsant effects are mediated by the inhibi- tion of Ca**-dependent release of GHB. In addition, the opiate receptor antagonist naloxone inhibits the GHB-induced cGMP increase* and attenuates the abnormal EEG activiv’. Taking into account the fact that GHB administration causes an incrrasc in dyno % hin level in the hippocampus , a region rich !n p-receptors, and that administration to the rat of certain opiates induces epileptic seizures which are antagonized by both valproate and etho- suximide*s, it could be suggested that these anticonvulsants act via a GHB-ergic mechanism which is linked to the endogenous opioid GHB administration to animals most often brings about epileptic system (Fig. 3). spiking activity. These pheno- mena may represent functional overload of synapses which release GHB. There is much sup- port for the existence of such synapses in the CNS. Valproate and ethosuximide modify the characteristics and functions of this group of synapses by inhibit- ~~ ~~~c~i~~~~*~~d~~e~~~n~ depolarization-induced release. Increases in cGMP brought about by GHB are blocked by valproate, ethosuximide and also by nalox- one. These three drugs antagonize the epileptogenic activity of GHB. The effect of naloxone may indi- cate that endorphins participate in the aetiology of this epileptic phenomenon. This biochemical mechanism could constitute it model for anticonvulsant agents. It would thus be of interest to synthesize new molecules which either reduce synaptic release of GHB or are antagonists at its receptor sites.
`
`19
`
`McLean, M. and McDonald, L. R. (1986) 1.
`Pharmacof.
`Exp.
`
`Ther. 237,1001-1011 20 Snead, 0. C., Bearden, L. J. and Pegram, V. (1980)
`
`Neurophannacology
`
`19,47-52
`
`21
`
`Rumigny, J. F., Maitre, M., Cash, C. and Mandel, P. (19801
`FEBS
`Lett.
`117,
`
`lll- 116 22 Vayer, I’., Schmitt, M., Bourguignon, J. J,, Mandel, P. and Maitre, M. (1985)
`
`FEBS
`
`Lett.
`
`190,
`
`55-60
`
`23
`
`Kaufman, E. and Nelson, Tb. (1987) J. Neurochem. 48,1935-1941 24 Vaver. P.. Charlier. B.. Mandel. I’. and M&r& h. (1987j J: Neurochem. 49, 1022-1024 25 Sneed, 0. C., Bearden, L. J and Pegram, V. (1980) Neuropharmacoh$y 19,4?-52 26 Snead, 0. C. and Bearden, L. J. (1980) Neurology 30,832-838 27 Lason, W., Przewlocka, B. and Przew- locka, R. (1983) Life Sci. 33,59%02 28 Snead, 0. C. and Bearden, L. J. (1982)
`
`Neuropharmacofogy
`
`21,1137-1144
`
`Transmembrane
`
`signalling Single copies of this centrefold can be purchased from our Cambridge office. See page 126.
`
`Acknowledgement
`
`Supported by grant from DRET (no. 85/1200).
`
`References
`1
`Vayer, I’., Mandel, I’. and Maitre, M. (1987) Life Sci. 41,1547-1557 2 Snead, 0. C. (1987) J.
`
`Neur~~he~n.
`
`PAR1012
`IPR of U.S. Patent No. 8,772,306
`Page 3 of 3
`
`TIPS -
`ethosuxlmlde
`\a/
`GHB release
`(endo) from
`+epileptlc disorders