Electrophysiological Properties

A vast number of both in vitro and in vivo electrophysiological studies in rodents has consistently shown an absence of effect of LEV on normal neuronal responses and neuronal characteristics.⁹ This contrasts several other studies reporting that LEV exerts a preferential action against hypersynchronization of epileptiform activity.

It has been observed that LEV differs from other AEDs by its ability to reduce increases in the amplitude of evoked population spikes, reflecting hypersynchronization, in rat hippocampal slices expressing epileptiform activity due to perfusion with high K⁺/low Ca²⁺.¹⁰ Further exploration with simultaneous dual extra- and intracellular recordings in the same model showed that LEV was the only AED to decrease the number of population spikes per extracellular response, without altering the number of action potentials per intracellular burst.¹¹ These results suggest that LEV may counteract the transition from interictal to ictal activity. This could explain both its absence of activity against acute seizures induced by immediate, ictal activity in normal animals as well as its selective seizure protection, and low induction of adverse effects in animals with acquired and genetic epilepsy.²

Conventional AED Mechanisms

The antiepileptic action of AEDs traditionally relates to a primary action on one or more of three main mechanisms. These consist of facilitation of GABA_A/BZ receptors; inhibition of voltage-gated Na⁺ channels, and inhibition of low voltage-gated (T-type) Ca²⁺ channels. Numerous studies have failed to show a direct interaction of LEV with any of these three mechanisms.⁹ This confirms that LEV’s unique profile in epilepsy pharmacology must reflect a novel mechanism of action.

Other Mechanisms

It has repeatedly been observed that LEV possesses an ability to inhibit AMPA-gated currents in rat hippocampal and cortical neurons.^12,13 This effect only becomes significant at concentrations above therapeutic relevance and can therefore not be expected to contribute to LEV’s antiepileptic action. However, other studies conducted at therapeutically relevant concentrations have discovered two novel cellular effects that probably contribute to LEV’s unique mechanism of action.

LEV was shown to differ from other AEDs by an ability to reverse the inhibition of Zn²⁺ and β-carbolines of both GABA-gated currents in rat hippocampal and dentate granule neurons and glycine-gated currents in spinal cord neurons (Table 3-1).¹⁴ This distinguishes LEV from other AEDs and associates it with a potentially novel mechanism, in particular when viewed in the context of the “sprouted mossy fiber/Zn²⁺-sensitive GABA_A receptor” hypothesis.¹⁵ Indeed, it has been proposed that epileptogenesis involves alterations in both the subunit composition of the GABA_A receptor as well as mossy fiber sprouting from dentate granule cells with Zn²⁺ containing terminals. This is believed to induce a vicious circle of disinhibition in hippocampus resulting in epileptic discharges—a condition that LEV may counteract.

TABLE 3–1 Levetiracetam, Brivaracetam, and Seletracetam: Mechanisms of Action

LEV was also been shown to reduce high-voltage gated Ca²⁺ currents.^16,17 This effect appears to relate to a selective modulation of N-type Ca²⁺ channels.¹⁸ Furthermore, several studies have reported on LEV’s ability to reduce both ryanodine and IP₃-receptor mediated Ca²⁺ release from the endoplasmic reticulum.^19,20 Taken together, these results suggest a novel effect of LEV on Ca²⁺ transients by a dual action on both N-type Ca²⁺ channels, incorporated in the plasma membrane, as well as on intraneuronal Ca²⁺ stores.

LEV’s ability to oppose Zn²⁺ inhibition of GABA- and glycine-gated currents and on Ca²⁺ transients are only of a modest magnitude. Thus, although novel, these mechanisms cannot constitute the primary antiepileptic mechanism of action of LEV. This appears, instead, to relate to LEV’s binding to the synaptic vesicle protein 2A.

Synaptic Vesicle Protein 2A

LEV (10 µM) has not been found to displace radioligands specific for a variety of receptors, ion channel proteins, reuptake sites, and second messenger systems.²¹ This contrasts to the observation of a specific binding site for ³H-LEV in rat brain membranes, which has become known as the Levetiracetam Binding Site (LBS).²¹ LEV was found to bind saturably, reversibly, and stereospecifically to LBS. A strong correlation also existed between the affinity of a series of LEV analogs to the LBS and their seizure protection in epilepsy models.²¹ This suggests an important functional role for LBS in the antiepileptic mechanism of LEV and provided a strong rationale to identify its molecular nature. Approximately 10 years after the discovery of LBS, it was finally documented that synaptic vesicle protein 2A (SV2A) is the molecular correlate to LBS.²²

SV2 is a 12-transmembrane protein incorporated in the membrane of synaptic vesicles, present in the presynaptic terminal. It exists in three isoforms, SV2A, SV2B, and SV2C, of which SV2A is the most widely distributed in the brain and also present on many neuroendocrine cells.²³ The role of SV2 in the modulation of synaptic events remains obscure, but it is presumed to exert a modulatory effect on maturation and/or fusion of vesicles with the plasma membrane of the presynaptic terminal.^23,24 Further evidence that the SV2A isoform has an impact on neurotransmission is derived from studies on animals lacking SV2A. These have shown that SV2A homozygous knockout (KO) mice express a lethal seizure phenotype,²³ and that SV2A heterozygous KO mice reveal accelerated kindling acquisition.²⁵ This supports the theory that SV2A has an important role in the control of vesicle exocytosis and may be involved in the pathophysiology of epilepsy.

LEV has been documented to bind to SV2A expressed in fibroblasts (Table 3-1) but reveals no significant binding to SV2B or SV2C.²² Binding of ³H-LEV to brain membranes and purified synaptic vesicles from SV2A KO mice was absent, further supporting that SV2A is the molecular correlate of LBS (Figure 3-2). A strong correlation for LEV analogs was confirmed between their affinity for SV2A and LBS as well as between their affinity for SV2A and seizure protection in animal models of epilepsy (Figure 3-3). No other AED tested, up to 100 µM, revealed any significant affinity for SV2A.

Figure 3–2 Binding of the tritiated levetiracetam analog [³H]ucb 30889 to WT and KO brain membranes and synaptic vesicles.A, Western blot of brain membranes from WT and homozygous KO mice probed with an anti-SV2 monoclonal antibody (cross-reactive to all isoforms) or with an anti-SV2A-specific polyclonal antibody. Lanes 1, WT; lanes 2, SV2A^-/- KO; lanes 3, SV2B^-/- KO; lanes 4, SV2A^-/-/B^-/- double KO. B, Binding of [³H]ucb 30889 to brain membranes from WT, SV2A^-/-, SV2B^-/-, and SV2A^-/-/SV2B^-/- KO mice. Binding is observed only to membranes from animals expressing SV2A. , [³H]ucb 30889 alone; , [³H]ucb 30889 plus 1 mM levetiracetam. Error bars are the SD of experiments performed with five WT brains and four KO brains, with three replicates within each experiment. C, Purification of synaptic vesicles enriched for the synaptic vesicle proteins and levetiracetam binding. Shown are blots of mouse brain homogenate (H), crude synaptosomes (P2), plasma and heavy membranes (LP1), and synaptic vesicles (LP2) (2 μg of each fraction) that were probed for the synaptic vesicle proteins SV2A (Upper) and synaptophysin (Lower). The synaptic vesicle fraction from WT animals displays enrichment of both synaptic vesicle proteins and LEV-binding proteins, whereas material from SV2A KOs shows enrichment of synaptophysin only. D, Binding to the different fractions using [³H]ucb 30889 shows significant binding only to the WT LP2 fraction, containing SV2A-rich synaptic vesicles. Shown are [³H]ucb 30889 alone (open bars) and [³H]ucb 30889 plus 1 mM levetiracetam (filled bars). Shown are representative examples of two experiments. Error bars are the SD of two replicates.

Reprinted with permission from Lynch, B et al. The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam. PNAS. 2004;101(26):9861-9866, © 2004 National Academy of Sciences, U.S.A.