The effects of stiripentol on GABA_A receptors

SUMMARY

The anti-convulsant Stiripentol (Diacomit^™) has been shown to have a positive impact on control of seizures for many patients with Dravet Syndrome. As with most anti-epileptic drugs, stiripentol has multiple mechanisms of action. Its direct anti-convulsant activity is likely due to enhancement of inhibitory, GABAergic neurotransmission. Stiripentol was shown to increase the activity of both neuronal and recombinant GABA_A receptors at clinically relevant concentrations. At recombinant receptors, stiripentol was found to act through a unique site in a subunit-dependent manner. Positive modulation by stiripentol was most effective at GABA_A receptors containing an α3 subunit. The expression of the α3 subunit is developmentally regulated, with highest levels in the immature brain. This subunit selectivity may explain the greater clinical efficacy of stiripentol in childhood-onset epilepsies, including Dravet Syndrome.

Stiripentol (STP) is a novel anti-epileptic drug (AED) structurally unrelated to other anticonvulsants used clinically to treat seizure disorders (Figure 1) (Trojnar et al., 2005). It has received orphan drug status as adjunctive therapy for the treatment of severe myoclonic epilepsy in infancy (SMEI, Dravet Syndrome) and can be effective in reduction of pharmacoresistant seizures and status epilepticus. STP has both direct and indirect anticonvulsant actions.

Figure 1. Structure of Stiripentol.

Stiripentol (4,4-dimethyl-1-[3,4-(methylene dioxy)-phenyl]-1-penten-3-ol) is structurally unrelated to other clinically available anti-convulsant drugs.

Stiripentol inhibits metabolism of other anti-convulsant drugs

STP has been tested for decades in combination with other AEDs because of its inhibitory action on metabolic enzymes. STP inhibits a variety of hepatic cytochrome P450 enzymes, with high activity at human CYP1A2, CYP3A4 and CYP2C19 (Tran et al., 1997). As a result, it increases the peak concentrations and duration of action of several commonly used AEDs, including phenobarbital, clobazam, carbamazepine, phenytoin and valproate (Trojnar et al., 2005; Landmark and Johannessen, 2008). For patients with Dravet syndrome, STP is commonly combined with clobazam. Evidence suggests that much of the anti-convulsant activity of clobazam is mediated through its active metabolite, norclobazam (N-desmethylclobazam) (Kinoshita et al., 2007). While co-administration with STP increases the concentration of both clobazam and norclobazam, it has a much greater impact on norclobazam levels (Giraud et al., 2006). Therefore, some of the improved efficacy seen with the combination of STP and clobazam may be due to increased levels of norclobazam. In addition to its effects on drug metabolism, there is strong evidence that STP alone is directly anti-convulsant. STP monotherapy reduces seizure severity in a variety of rodent models and in a primate model of focal epilepsy (Trojnar et al. 2005). This suggested that STP has additional effects on seizure activity independent of alterations in drug metabolism. Subsequent studies have shown that STP acts directly on GABA_A receptors (GABA_ARs) as a positive allosteric modulator.

Stiripentol enhances GABAergic neurotransmission

The GABA_ARs are ligand-gated chloride channels responsible for fast inhibitory neurotransmission. They are targets for many commonly used anti-convulsants, including benzodiazepines and barbiturates. Experimental evidence for a direct action of STP on the activity of GABA_ARs was first provided by Quilichini et al. (2006). Using a neonatal rat brain slice preparation they found that STP slowed the decay of GABAergic mIPSCs in hippocampal CA3 pyramidal cells. STP increased both the frequency and the duration of post-synaptic events, suggesting both pre- and post-synaptic effects. A direct modulation of GABA_ARs was confirmed with single-channel recordings, which showed that STP increased the mean open time of GABA-activated channels.

Stiripentol has subunit-dependent effects on recombinant GABA_A receptors

The finding that STP interacts with post-synaptic GABA_ARs in hippocampal neurons was extended by studies of recombinant receptors (Fisher, 2009). Neuronal GABA_ARs can have a large degree of structural heterogeneity due to variations in subunit composition. At least 16 different GABA_AR subunits have been found in mammalian brain and the composition of the receptor has a significant impact on its functional and pharmacological properties (Korpi et al., 2002).

STP was found to positively modulate all recombinant GABA_ARs tested, but had its greatest effect when the receptor contained an α3 subunit. Unlike the benzodiazepines, STP did not require presence of a γ subunit and was a strong modulator of δ-containing receptors. STP was shown to enhance GABA_AR activity by increasing sensitivity to GABA without increasing the peak current to maximally effective GABA concentrations. This mechanism is similar to that of benzodiazepine-site agonists, but the STP site is clearly distinct from other well-known GABA_AR modulators. The activity of STP is not blocked by antagonists of the benzodiazepine or neurosteroid sites (Quilichini et al., 2006; Fisher, 2009) nor is it influenced by mutations that disrupt loreclezole or neurosteroid activity (Fisher, 2009). Co-administration studies suggest that STP acts independently and additively with benzodiazepines and neurosteroids, though it is not able to further enhance modulation by pentobarbital (Quilichini et al., 2006 and Fisher, 2009). STP has a unique pattern of subunit dependence unlike any other GABA_AR modulator. Its particularly high activity at α3- and δ-containing receptors could have important implications for its effect on neuronal activity and its clinical usefulness.

Characteristics of the α3 subunit of GABA_A receptors

Expression of the α3 subunit is strongly regulated by development. mRNA for the α3 is found at relatively high levels in most brain regions of the embryonic and newborn mammal (Laurie et al., 1992). As maturation progresses, its expression becomes more restricted and it is produced primarily by neurons in the cortex and thalamus of the adult brain. However, higher production of the α3 subunit has been found in adult animals that have a greater susceptibility to seizures (Poulter et al., 1999). GABA_ARs containing the α3 subunit have been suggested to play an important role in normal cortical development, and in adults may mediate some anxiolytic and anticonvulsant effects of GABA-modulating drugs (Sohal et al., 2003; Yee et al., 2005; Dias et al., 2005).

Characteristics of the δ subunit of GABA_A receptors

GABA_ARs containing the δ subunit represent a unique population of receptors (Belelli et al., 2009). These receptors are exclusively located in extra-synaptic regions, and contribute to the long-lasting tonic current observed in the hippocampus, cerebellum and thalamus in response to the low ambient levels of GABA. The δ subunit confers functional properties such as high agonist sensitivity and minimal desensitization that make them well-suited for this role. The δ-containing receptors also have unique pharmacological properties. In particular, they are insensitive to positive modulation by the benzodiazepine class of AEDs. Therefore, drugs such as STP that enhance the activity of these receptors may retain effectiveness in patient populations that are pharmacoresistant to benzodiazepines.

Summary

The studies of neuronal and recombinant GABA_ARs confirm that STP can have a direct action on neurotransmission, supporting its anti-convulsant action in animal models of acute seizures. The preferential enhancement of α3-containing receptors provides a plausible mechanism for the observation that STP is very effective in treating childhood seizures, despite its lower clinical utility in adults. These findings reinforce the need to consider the unique characteristics of the immature, developing brain when designing therapies to treat neurological disorders of infants and children.