- Top of page
Losigamone ((±)-5(R,S),α(S,R)-5-(2-chlorophenyl)hydroxymethyl)-4-methoxy(5H)-furanone) is a novel anticonvulsant the mechanism action of which is not known. It is undergoing Phase II and III clinical trials in patients with partial and secondary generalized seizures. Losigamone belongs to the group of β-methoxy-butenolides which is found in a number of natural substances (Stein, 1995). Losigamone inhibits the tonic hindleg extension produced by electroshock, pentylenetetrazol (PTZ), bicuculline, nicotine and 4-aminopyridine (Stein, 1995). The clonic components of seizures induced by PTZ, bicuculline and picrotoxin are also suppressed by losigamone but it has no effect on the hindleg extension caused by strychnine and picrotoxin (PTX) or the clonic seizures provoked by N-methyl-d-aspartate (NMDA) (Stein, 1995). In the maximal electroshock test (MES) losigamone is more potent than phenytoin and valproate; and in the PTZ test it is more effective than ethosuximide and valproate. Toxicity studies have not produced any significant abnormalities and it does not appear to be teratogenic in animals (Stein, 1995).
In in vitro studies losigamone has been shown to reduce the frequency of the spontaneous epileptiform discharges produced by both PTX and perfusion with low-magnesium and low-calcium containing aCSF (Kohr & Heinemann, 1990a; Leschinger et al., 1993). It has also been shown to block epileptiform discharges in areas CA1 and CA3 of the hippocampus and in the entorhinal cortex (Kohr & Heinemann, 1990b). Losigamone produces a concentration-dependent increase in chloride uptake in cultured spinal cord neurones and potentiates the effect of exogenous γ-aminobutyric acid (GABA; Dimpfel et al., 1995). However, it does not affect [3H]-flunitrazepam or [3H]-GABA binding (Dimpfel et al., 1995). Losigamone has been shown, in studies on sustained repetitive-firing in hippocampal-entorhinal cortex slices, to reduce firing (Schmitz et al., 1995) as well as reducing excitatory postsynaptic potential (e.p.s.p.) amplitudes, while monosynaptic fast and slow inhibitory postsynaptic potential (i.p.s.ps) were unaffected (Schmitz et al., 1995). These results suggest that the drug has a primary mechanism of action on the neuronal membrane. However, unlike phenytoin and carbamazepine which also suppress repetitive firing, losigamone inhibits late recurrent discharges and these are readily inhibited by NMDA antagonists. A study of losigamone on 4-aminopyridine (4-AP)-induced epileptiform activity in the hippocampus showed that losigamone, like other anticonvulsants, partially reversed 4-AP excitation (Yonekawa et al., 1995). The same group also studied the effect of anticonvulsants on 4-AP-induced de novo synthesis of amino acid neurotransmitters in rat hippocampus and, in common with other drugs that block use-dependent, voltage-sensitive sodium channels such as phenytoin, carbamazepine and lamotrigine, losigamone inhibited 4-AP-induced synthesis of glutamate, aspartate and GABA (Kaptenovic et al., 1995).
Genetically epilepsy-prone DBA/2 mice are susceptible to sound-induced seizures (Jobe et al., 1991) and cortical wedges from these mice, especially in the absence of Mg2+ in the extracellular perfusate, exhibit spontaneous depolarizations (Hu & Davies, 1995). This present study investigated the effect of a racemic mixture of losigamone on spontaneous and NMDA-and α-amino-3-hydroxy-5-methyl-4-isoxazolapropionate (AMPA)-induced depolarizations in cortical wedges prepared from DBA/2 mice. Also the effect of losigamone on potassium-and veratridine-induced release of endogenous amino acids from cortical slices prepared from BALB/c mice was examined.
- Top of page
The results presented here show that losigamone significantly reduced the frequency of spontaneous depolarizations at concentrations of 100 μm and above, and the frequency of associated afterpotentials at 25 μm and above, in the cortical wedge preparation from DBA/2 mice. It also had an inhibitory effect on NMDA-induced depolarizations but had no effect on AMPA-induced depolarizations.
DBA/2 mice are genetically epilepsy-prone and this behaviour is age related, in that mice of 20–30 days of age are far more susceptible to seizures than younger or older mice. In our colony, 96% of mice at this age respond to a 110 dB sound with characteristic wild-running and tonic clonic seizures (Abila et al., 1993). As we have previously shown (Hu & Davies, 1995), 90% of slices prepared from animals aged between 20–30 days exhibit spontaneous depolarizations in magnesium-free medium. The role of NMDA receptors in seizure initiation and propagation is well recognized (Meldrum, 1992) and potential anticonvulsant compounds, acting on diverse sites of the NMDA receptor, have been shown to reduce burst frequency and the number of afterpotentials in the rat cortical wedge preparation (Aram et al., 1989). This is suggestive that NMDA receptor complex activation is involved in these epileptiform events in this in vitro preparation. Anticonvulsant compounds acting at other sites, such as σ ligands and GABA agonists have also been shown to be inhibitory in the cortical wedge preparation (Horne et al., 1986; Palmer et al., 1992).
The inhibitory effect of losigamone on spontaneous depolarizations, associated afterpotentials and NMDA-induced depolarizations shown in this study is suggestive of NMDA-receptor antagonism. It is not possible to ascertain from the above results the site of action of losigamone on the NMDA receptor, as drugs acting at different sites on the NMDA receptor would be capable of reducing spontaneous and NMDA-induced depolarizations. These present results do not support those of Stein (1995), who showed no effect of losigamone on NMDA-induced convulsions in mice.
In addition, this study showed that losigamone reduced veratridine-induced release of glutamate at concentrations of 100 μm and above, and potassium-induced release of glutamate and potassium- and veratridine-induced release of aspartate at concentrations of 200 μm. As previously shown, potassium-stimulated release is calcium-dependent while veratridine-stimulated release is only partially affected by removal of calcium from the medium (Srinivasan et al., 1995).
The mechanism by which an increase in extracellular potassium stimulates release of excitatory amino acids is secondary to changes in the transmembrane potential, resulting in the opening of voltage-sensitive calcium channels. The release of neurotransmitters in response to potassium does not involve sodium channels as tetrodotoxin, a potent sodium channel blocker, has been shown to be ineffective in inhibiting this release (Dickie & Davies, 1992). Activation of the NMDA receptor by the endogenous ligand initiates a positive feedback loop occurring at the synaptic level, which acts on the presynaptic terminal to increase the release of transmitter. There are various modulators which have been shown to be involved in this positive feedback response, including nitric oxide (Rowley et al., 1993; Montague et al., 1994) and arachidonic acid (Dickie et al., 1994). Drugs which are NMDA receptor antagonists may prevent this positive feedback resulting in reduced neurotransmitter release and, conversely, NMDA has been shown to stimulate glutamate release from mouse cortical slices (Rowley et al., 1993). The glutamate released from the cortical slice preparation probably comes from the glutamatergic neurones, which form association and commissural fibres, and the dendrites of these neurones possess NMDA receptors (Huntley et al., 1994). If activation of these receptors is blocked by an NMDA antagonist, as is probably the case with losigamone, then augmentation of glutamate release by NMDA receptor stimulation will not occur. The differences in concentration of losigamone between that required to reduce potassium-stimulated release of glutamate and that which reduced NMDA-induced depolarizations is probably due to the nature of the stimulation. We have previously found (Hu & Davies, 1995) that exogenous application of NMDA was reduced by remacemide at lower concentrations than was potassium-stimulated glutamate release (Srinivasan et al., 1995), which, by nature of the methodology, is through endogenous mechanisms.
Veratridine releases neurotransmitters by preventing the inactivation of sodium channels and tetrodotoxin, which blocks sodium channels, prevents this release (Levi et al., 1980; Minchin, 1980). Drugs such as lamotrigine, which inhibit veratridine-stimulated release of excitatory amino acids but have no effect on potassium-stimulated release, are therefore thought to act by maintaining the inactivation of sodium channels (Leach et al., 1986). Recently published results are compatible with the observation that losigamone is a sodium channel blocker (Schmitz et al., 1995). Losigamone has been shown in studies on sustained repetitive-firing in hippocampal-entorhinal cortical slices (a test which involves the activation of voltage-operated sodium channels) to reduce firing (Schmitz et al., 1995). In addition, losigamone reduced e.p.s.p. amplitudes while monosynaptic fast and slow i.p.s.ps were unaffected. These results suggest that the drug has a primary mechanism of action on the neuronal membrane. However, unlike phenytoin and carbamazepine which also suppress repetitive firing, losigamone inhibits late recurrent discharges and these are readily inhibited by NMDA antagonists. Losigamone, in common with other drugs that block use-dependent voltagesensitive sodium channels, such as phenytoin, carbamazepine and lamotrigine, inhibited 4-AP-induced synthesis of glutamate, aspartate and GABA (Kaptenovic et al., 1995). The inhibitory effect of losigamone on veratridine-induced release of glutamate and aspartate could therefore be secondary to sodium channel blockade. However, the effect of losigamone in reducing potassium-stimulated release of glutamate and aspartate, is not a sodium channel effect as TTX does not reduce release. The action of losigamone probably involves NMDA receptor antagonism as we have previously shown that dizocilpine (MK-801) reduced potassium-stimulated release of glutamate (Srinivasan et al., 1995).
The NMDA receptor antagonism and inhibition of excitatory amino acid release demonstrated here could be relevant to the anticonvulsant effect of losigamone.
We are grateful to Dr S.S. Chatterjee of Willmar Schwabe Arzneimittel for the gift of losigamone.