Certain steroid hormone metabolites that have activity as modulators of GABA_A receptors but lack conventional hormonal effects—including allopregnanolone and allotetrahydrodeoxycorticosterone—are synthesized within the brain, predominantly in principle (excitatory) neurons, and also in peripheral tissues. At low concentrations, such neurosteroids potentiate GABA_A receptor currents, whereas at higher concentrations they directly activate the receptor; large magnitude effects occur on nonsynaptic δ subunit-containing GABA_A receptors that mediate tonic currents. GABA_A receptor modulatory neurosteroids confer seizure protection in diverse animal models, without tolerance during chronic administration. Endogenous neurosteroids may play a role in catamenial epilepsy, stress-induced changes in seizure susceptibility, temporal lobe epilepsy, and alcohol withdrawal seizures. Moreover, neurosteroid replacement with natural or synthetic neurosteroids may be useful in these conditions and more generally in the treatment of partial seizures. Ganaxolone, the synthetic 3β-methyl analog of allopregnanolone, has been evaluated in clinical trials for the treatment of epilepsy. It appears to be an efficacious, well-tolerated and safe treatment for partial seizures. Neurosteroids and analogs such as ganaxolone show promise in the treatment of diverse forms of epilepsy.

INTRODUCTION

The term ‘neurosteroid’ was originally coined in 1981 by the French endocrinologist Étienne-Émile Baulieu to refer to steroids that are synthesized de novo in the nervous system from cholesterol independently of the peripheral steroidogenic endocrine glands.¹ Paul and Purdy then characterized ‘neuroactive steroids’ as “natural or synthetic steroids that rapidly alter the excitability of neurons by binding to membrane-bound receptors such as those for inhibitory and (or) excitatory neurotransmitters.”² This chapter is concerned with the anticonvulsant and antiepileptogenic properties of neurosteroid-related steroid molecules (i.e., related to endogenously synthesized steroids) that meet the Paul and Purdy definition of neuroactive steroids by virtue of their pharmacological actions as positive allosteric modulators of GABA_A receptors. The effect of these steroids on GABA_A receptors occurs through a direct action on GABA_A receptors and is not related to interactions with classical steroid hormone receptors that regulate gene transcription. Indeed, GABA_A receptor modulatory neurosteroids are not themselves active at intracellular steroid receptors. We will make scant mention of other endogenous neurosteroids that exert other types of pharmacological actions, such as inhibition of GABA_A receptors or effects on excitatory amino acid receptors (pregnenolone sulfate is an example of such a steroid) although it is conceivable that neurosteroids with such actions could play a role in regulating seizure susceptibility.

It has been known since the 1940s from the pioneering work of Hans Selye that naturally occurring steroids such as the ovarian steroid progesterone and the adrenal steroid deoxycorticosterone (DOC) can exert anesthetic and anticonvulsant actions.³ Recognizing that some steroids could produce such acute central nervous system effects, research at the pharmaceutical company Glaxo identified the synthetic steroid alphaxolone as having anesthetic properties. In the early 1970s, alphaxolone was marketed as a component of the intravenous anesthetic agent Althesin, which also included the less potent anesthetic steroid alphadalone acetate that was said to increase the solubility of alphaxolone.⁴ Several years later, the mechanism of action of alphaxolone was defined: it was found to enhance synaptic inhibition via an action on GABA_A receptors.^5,6 A major advance occurred when naturally occurring metabolites of progesterone and DOC were also found to enhance and directly activate GABA_A receptors.⁷ It was speculated that the anesthetic and hypnotic properties of progesterone and DOC known since the time of Selye were due to their conversion in the body to these metabolites, respectively, allopregnanolone (3α-hydroxy-5α-pregnane-20-one) and allotetrahydrodeoxycorticosterone (3α,21-dihydroxy-5α-pregnan-20-one; THDOC). At the time, it was recognized that the enzymes required for the conversion of the steroid hormone precursors to their active A-ring reduced metabolites are present in brain so that part of the synthesis of the GABA_A receptor active steroids could occur locally. Therefore, the neuroactive steroids allopregnanolone and THDOC came to be referred to as neurosteroids even though it was not believed at the time that their synthesis occurred independently of peripherally synthesized precursor steroid hormones. We now know that all of the enzymes required for the synthesis of the GABA_A receptor active steroids from cholesterol are present in the brain.⁸ It is well recognized that these steroids readily cross the blood-brain barrier. While it is likely that locally synthesized GABA_A receptor active steroids play a role in modulating circuit excitability, there is little information on the relative importance of de novo local synthesis versus peripheral production of either the active GABA_A receptor modulatory steroids (e.g., allopregnanolone or THDOC) or their precursor hormones (e.g., progesterone or DOC) which are converted locally by brain 5α-reductase and 3α-hydroxysteroidoxidoreductase (3α-HSOR). Nevertheless, it is common to refer to GABA_A receptor modulatory steroids such as allopregnanolone and THDOC as neurosteroids and we will follow that practice here. This chapter reviews the potential roles of such neurosteroids as endogenous modulators of seizure susceptibility and also the more limited evidence that they can under certain circumstances influence epileptogenesis (transformation of the brain to an epileptic state). The discussion of these topics serves as a prelude to the main objective of this chapter, which is to review the evidence supporting the utility of exogenously administered neurosteroid-related agents in the treatment of epilepsy.

DIVERSITY OF NEUROSTEROIDS AND THEIR BIOSYNTHESIS

A variety of GABA_A receptor modulatory neurosteroids are known to be synthesized endogenously (Figures 1 and 2). The best recognized of these are the pregnane neurosteroids allopregnanolone and THDOC that are produced via sequential A-ring reduction of the steroid hormones progesterone and its 21-hydroxylated derivative deoxycorticosterone by 5α-reductase and 3α-HSOR isoenzymes. In the periphery, the steroid precursors are mainly synthesized in the gonads, adrenal gland, and feto-placental unit, but as noted above, synthesis of both of these neurosteroids likely occurs in the brain from cholesterol or from peripherally derived intermediates including the steroid hormone precursors. A-ring reduction can also occur in peripheral tissues such as reproductive endocrine tissues, liver, and skin that are rich in the two reducing activities.⁸ Since neurosteroids are highly lipophilic and can readily cross the blood-brain barrier, neurosteroids synthesized in peripheral tissues accumulate in the brain and can influence brain function.

Figure 1

Biochemical pathways of neurosteroid biosynthesis. Cholesterol is trafficked by StAR (sterodogenic acute regulatory protein) and TSPO (translocator protein 18 kDa) to the inner mitochondrial membrane where it is converted to pregnenolone by P450sc (cytochrome (more...)

Figure 2

General scheme of peripheral steroidogenesis showing side pathways for the production of known neurosteroids. Steroids known to act on GABA_A receptors in a similar fashion to the prototypic neurosteroids allopregnanolone and allotetrahydrodeoxycorticosterone (more...)

The 5β-isomers of allopregnanolone and THDOC have GABA_A receptor modulatory activity that is only modestly less potent than the corresponding 5α-epimers.⁹ A steroid 5β-reductase enzyme is distributed widely in vertebrates, including in the gonads, liver and brain.¹⁰ However, whether progesterone or DOC is a substrate and the extent to which 5β-reduced epimers of allopregnanolone and THDOC are produced endogenously is unclear.

The androgenic steroid testosterone differs from progesterone by virtue of a 17-alcohol that replaces the 17-acetyl in progesterone. Testosterone is a substrate for both 5α-reductase and 5β-reductase isoenzymes. The product of 5α-reduction of testosterone, 5α-dihydrotestosterone, is hormonally more active than testostosterone itself. However, subsequent 3α-reduction leads to 5α-androstanediol (5α-androstane-3α,17β-diol), which is comparable in potency and efficacy to allopregnanolone as a GABA_A receptor positive modulator.¹¹ 5α- and 5β-Androstanediol are further metabolized by 17β-hydroxysteroid dehydrogenase to androsterone and etiocholanolone, respectively, which are considered the major excreted metabolites of testosterone. These compounds and their conjugates are present at high concentrations. Androsterone and etiocholanolone also have GABA_A receptor positive modulatory activity and represent endogenous neurosteroids.¹² Collectively, the various GABA_A receptor modulatory steroids that lack the pregnane 17β-ethyl moiety, such as 5α-androstanediol, androsterone and etiocholanolone can be considered androstane neurosteroids. Finally, substantial amounts of androstenol, the 16-unstaturated form of 5α-androstanediol, are present in mammals, including humans. This compound is considered to be a pheromone that increases sexual receptivity in pigs and possibly other species. Androstenol also is a GABA_A receptor positive modulator that has similar efficacy but is modestly less potent than allopregnanolone.¹³

PRODUCTION OF NEUROSTEROIDS IN THE BRAIN AND THEIR LOCALIZATION TO PRINCIPAL NEURONS

In addition to peripheral production, it is clear that neurosteroids can be formed from steroid hormone precursors (such as progesterone, DOC and perhaps testosterone) locally in the brain. 5α-Reductase activity has been identified in both neurons and glial cells in rodent and sheep brain, in regions, such as the neocortex and hippocampus, that are relevant to epilepsy.^14,15 3α-HSOR is also expressed widely in the brain.¹⁶ In humans, both enzymes have been found in neocortex and hippocampus.^17–19 Thus, it is likely that neurosteroids can be formed from their parent steroid hormone precursors directly in the brain. Steroid precursors readily enter the brain so that pools of peripherally synthesized precursors are available for local neurosteroid biosynthesis. In peripheral tissues 5α-reductase is believed to be the rate-limiting step in the production of neurosteroids because 3α-HSOR is a more ubiquitous enzyme; the same situation likely applies in brain.

It is likely that neurosteroids can be produced locally in brain not only from their steroid hormone precursors but also from more elementary steroid precursors such as cholesterol or pregnenolone. The rate-limiting and initial step in steroidogenesis is the conversion of cholesterol to pregnenolone by the mitochondrial enzyme P450scc (cytochrome P450 cholesterol side-chain cleavage enzyme; CYP11A). Access of cholesterol to P450scc requires StAR (steroidogenic acute regulatory protein), which functions to transfer cholesterol from the outer mitochondrial membrane to the inner membrane where P450scc is located.²⁰ Translocator protein (18 kD) (TSPO), formerly called peripheral or mitochondrial benzodiazepine receptor, likely functions as a complex with StAR.^21,22 Both proteins are expressed widely in peripheral tissues and in the brain. Activation of TSPO by certain ligands facilitates the intramitochondrial flux of cholesterol and thereby increases the availability of cholesterol to P450scc, enhancing pregnenolone synthesis and ultimately neurosteroid production.^23,24 The observation that TSPO ligands enhance neurosteroid production not only confirms the key role of TSPO in neurosteroidogenesis, but suggests that such ligands may have therapeutic utility as an alternative to exogenously administered neurosteroids in situations where it is desirable to increase brain neurosteroids.²⁵

In addition to P450scc, 3β-hydroxysteroid dehydrogenase, an enzyme required for the conversion of pregnenolone to progesterone, has been demonstrated in the brain.²⁶ Thus, the enzymes necessary for in situ synthesis of progesterone from cholesterol are present in the brain.

In the adrenal, P450c21 (cytochrome P450 21-hydroxylase) converts progesterone to DOC, which is the precursor for the neurosteroid THDOC. The brain also possesses 21-hydroxylase activity, but it expresses only very small amounts of P450c21 mRNA and protein. It appears that CYP2D isoforms, in particular CYP2D4, present in brain can 21-hydroxylate progesterone to form DOC, which can then be converted to THDOC.²⁷ In addition, allopregnanolone itself is a substrate for CYP2D4, so that in brain allopregnanolone may be converted directly to THDOC. Allopregnanolone and THDOC persist in the brain after adrenalectomy and gonadectomy or after pharmacological suppression of adrenal and gonadal steroid synthesis,^28,29 confirming that these two key neurosteroids can be synthesized independently of peripherally produced steroid hormone precursors. However, the regulatory mechanisms underlying neurosteroid biosynthesis in the brain remain unclear.

In studies with mouse and rat brain, in situ hybridization with mRNA probes to 5α-reductase and 3α-HSOR indicates that the two mRNAs colocalize to glutamatergic principal neurons and not GABAergic inhibitory neurons or glial cells within neocortex, hippocampus, amygdala and other brain regions.³⁰ Immunohistochemistry with an antiserum raised against allopregnanolone that also recognizes THDOC confirms that the neurosteroids are concentrated in principal neurons, predominantly in cell bodies and thick dendrites.³¹ The highly restricted distribution of neurosteroids to principal neurons suggests that they are mainly derived from local synthesis and not from the circulation, although it is clear that peripheral neurosteroids do readily cross the blood–brain barrier. It is remarkable that brain neurosteroids are localized to the neurons that contain their targets (GABA_A receptors). This observation is consistent with the notion that neurosteroids function in an autocrine fashion in which they reach their targets by lateral membrane diffusion.³²

NEUROSTEROID MODULATION OF GABA_A RECEPTORS

In electrophysiological studies, allopregnanolone and THDOC at aqueous concentrations in the range 10–1500 nM enhance the activation of GABA_A receptors by GABA.^9,33 At higher concentrations, the steroids directly activate the receptor in the absence of GABA. In addition, like other positive allosteric modulators of GABA_A receptors, neurosteroids exert allosteric effects on these receptors such that there is enhancement of the binding of [³H]flunitrazepam, a benzodiazepine receptor agonist, and [³H]muscimol, a specific GABA-site agonist; and inhibition of the binding of [³⁵S]t-butylbicycloorthobenzoate (TBPS), a cage convulsant and noncompetitive GABA_A receptor antagonist.^34,35 Neurosteroid enhancement of GABA_A receptors occurs through increases in both the channel open frequency and channel open duration.^36–38 Thus, neurosteroids greatly enhance the probability of GABA_A receptor chloride channel opening, thereby enhancing GABA_A receptor mediated inhibition.

The effects of neurosteroids on GABA_A receptors occurs by binding to discrete sites on the receptor-channel complex that are located within the transmembrane domains of the α- and β-subunits.³⁸ The binding sites for neurosteroids are distinct from the recognition sites for GABA, benzodiazepines and barbiturates. Although the exact location of neurosteroid binding has not been mapped, it has been proposed that there are two distinct sites for neurosteroids that act as positive modulators: one for allosteric enhancement of GABA and another for direct activation of the receptor.^37,38 Using site-directed mutagenesis, it has been shown that a highly conserved glutamine at position 241 in the M1 domain (toward the intracellular side) of the α-subunit plays a key role in neurosteroid modulation of GABA responses and is believed to contribute to the binding site for modulation.³⁹ Additional nearby residues in the M4 domain of the same α-subunit (tyrosine 410 and asparagines 407, which are located more toward the extracellular side) have also been proposed to contribute to the binding site. Other investigators have found that mutations in serine 240 and tryptophan 245 of the α-subunit interfere with neurosteroid potentiation.⁴⁰ Studies with structurally diverse steroids have led to the conclusion that the steroid binding pocket on the α-subunit is more correctly viewed as a “hydrophobic surface” that can accommodate steroid molecules of differerent structures.⁴⁰ Direct activation of the receptor, in contrast, has been proposed to be due to binding at a site on the interface between β and α subunits formed by a threonine at position 236 in the α-subunit and a tyrosine at position 284 in the β-subunit.³⁸ However, more recent models of the GABA_A receptor have questioned whether these residues reside at β subunit–α subunit interface.⁴¹ A photo-incorporable analog of the anesthetic etomidate appears to bind at the interface but binding of this ligand is not competitively inhibited by neurosteroids.⁴¹ In fact, neurosteroids (at concentrations that produce direct receptor activation) enhance binding, presumably due to allosteric effects transmitted upon interaction with a different site on the receptor. The newer topology models do not bring into proximity the residues in the β- and α-subunits proposed to constitute the site for direct activation. Therefore, at present, the location of this site is uncertain.

A range of steroid structures have activity as positive modulators of GABA_A receptors in line with the hydrophobic surface binding site model. Nevertheless, there are certain strict structural requirements for neurosteroid positive modulation. A hydrogen bond-donating 3α-hydroxy group on the steroid A-ring and a hydrogen bond accepting group (typically a keto moiety) on the D ring at either C20 of the pregnane steroid side chain or C17 of the androstane ring system are critical for positive modulatory activity at GABA_A receptors.^37,42 The orientation of the C5 hydrogen group only modestly influences potency.⁹

Studies with recombinant GABA_A receptor isoforms indicate that neurosteroids act on most subunit combinations.^37,43 This distinguishes neurosteroids from benzodiazepines, which only act on GABA_A receptors that contain γ2 subunits and do not contain α4 or α6 subunits. In general, the specific α-subunit type may influence neurosteroid efficacy, whereas the γ subunit type may affect both the efficacy and potency of neurosteroid modulation.³⁷

GABA is a relatively low-efficacy agonist of GABA_A receptors in which the δ subunit replaces the more common γ2 subunit, even though it binds with high affinity to such δ-subunit-containing receptors.⁴⁴ Neurosteroids therefore have an opportunity to markedly enhance the current generated by δ-subunit-containing GABA_A receptors even in the presence of saturating GABA concentrations. Consequently, GABA_A receptors that contain the δ subunit are highly sensitive to neurosteroid-induced potentiation of GABA responses^45,46 and mice lacking δ-subunits show drastically reduced sensitivity to neurosteroids.^47–49 GABA_A receptors containing δ-subunits exhibit low desensitization and they are located nonsynaptically (perisynaptically/extrasynaptically) since the γ2 subunit is required for synaptic targeting. These properties cause them to be prime candidates for mediating ‘tonic’ GABA_A receptor current that is activated by ambient concentrations of GABA in the extracellular space. Ambient GABA is believed to result from spillover of synaptically-released GABA; concentrations would increase with high levels of activity of GABAergic interneurons as occurs during seizures. Tonic GABA_A receptor current causes a steady inhibition of neurons and reduces their excitability. Neurosteroids could therefore have a general role in setting the level of excitability and might specifically potentiate tonic inhibition during seizures when ambient GABA may rise. Overall, the robust effect of neurosteroids is likely to be due to their action on both synaptic and perisynaptic/extrasynaptic GABA_A receptors.

Although neurosteroids are viewed as high-potency modulators of GABA_A receptors since they are effective at concentrations in the mid-nanomolar to low micromolar range in aqueous solution, recent studies indicate that neurosteroid binding to the GABA_A receptor is actually of low affinity (K_d, ~1 mM).³² The high effective potency of neurosteroids results from partitioning of the lipophilic steroids within the plasma membrane, such that the concentrations presented to the receptor are orders of magnitude greater. Neurosteroids access the GABA_A receptor from the lipophilic plasma membrane. The non-specific accumulation and removal of the neurosteroids from the membrane are the major factors determining the rates of neurosteroid action when applied to cells via aqueous solution; rates of binding and unbinding to the receptor are only secondary factors.⁵⁰ It is noteworthy that intracellular delivery through the plasma membrane is compatible with the autocrine mechanism discussed above where the neurosteroids act on the GABA_A receptors in the same neurons in which they are produced.

As noted, at high concentrations (>10 μM), neurosteroids can directly receptor activate GABA_A channels in the absence of GABA.^9,51,52 In this respect, neurosteroids resemble barbiturates but not benzodiazepines.⁵³ Given the high concentrations required, whether direct actions are relevant to the role of endogenous neurosteroids or to the pharmacological actions of exogenously administered neurosteroid-related agents is not well understood.

ANTICONVULSANT AND ANTIEPILEPTOGENIC EFFECTS OF NEUROSTEROIDS

Exogenously administered neurosteroids, like other agents that act as positive GABA_A receptor modulators, exhibit broad-spectrum anticonvulsant effects in diverse rodent seizure models. They protect against seizures induced by GABA_A receptor antagonists including pentylenetetrazol (PTZ) and bicuculline and they are effective against pilocarpine-induced limbic seizures and seizures in kindled animals.^9,54–57 However, neurosteroids may exacerbate generalized absence seizures.^58,59 The potencies of neurosteroids in models where they confer seizure protection vary largely in accordance with their activities as positive allosteric modulators of GABA_A receptors. Thus, allopregnanolone has roughly equal potency to THDOC, but androstanediol, androsterone and etiocholanolone are somewhat less potent.^12,60,61 Like other anticonvulsant agents that act on GABA_A receptors, neurosteroids are inactive or only weakly active against electrically induced tonic extension seizures elicited according to the maximal electroshock (MES) protocol that is widely used for drug screening. However, they are active in the 6-Hz model in mice in which limbic-like seizure are induced by electrical stimulation of lower frequency and longer duration than in the MES test.⁶² In general, neurosteroids have comparable potencies in the 6-Hz and PTZ models. Neurosteroids are also highly effective in suppressing seizures due to withdrawal of GABA_A receptor modulator drugs including neurosteroids and benzodiazepines (diazepam), and also due to other types of agents such as ethanol, which may act in part through GABA_A receptors.^63–65 In constrast to benzodiazepines where utility in the chronic treatment of epilepsy is limited by tolerance, anticonvulsant tolerance is not obtained with neurosteroids.^66,67 Thus, neurosteroids have the potential to be used in the chronic treatment of epilepsy and this has been borne out in clinical trials (see below). The mechanisms responsible for tolerance to benzodiazepines are not known. However, factors such as uncoupling of the allosteric linkage between the GABA and benzodiazepine sites and changes in receptor subunit turnover with switching of subunits may be contributing mechanisms.⁶⁸ Neurosteroids do not act on the benzodiazepine site of GABA_A receptors, and they are able to modulate all isoforms of GABA_A receptors, even those that contain benzodiazepine-insensitive α4 and α6 subunits or do not include the obligatory γ2 subunit required for benzodiazepine-sensitivity. Thus, it is clear that neurosteroids can act on GABA_A receptors where the proposed benzodiazepine toleance mechanisms have been invoked. Surprisingly, while chronic neurosteroid exposure does not lead to anticonvulsant tolerance, chronic neurosteroid exposure does lead to tolerance to benzodiazepines.⁶⁷ Thus it appears that the same plastic changes that underlie benzodiazepine tolerance are brought into play by chronic neurosteroid exposure. However, neurosteroids, acting at distinct sites on GABA_A receptors and exhibiting effects on the full range of GABA_A receptor isoforms, do not exhibit anticonvulsant tolerance. Whether tolerance can occur to other pharmacological actions of neurosteroids remains to be determined.

The sulfated neurosteroids pregnenolone sulfate and dehydroepiandosterone sulfate, which act as GABA_A receptor antagonists, are proconvulsant when administered at high doses into the brain, producing seizures and status epilepticus.^69,70 Compelling evidence that such steroids exist endogenously in the brain is lacking and in any case it is unlikely that they exist at sufficiently high concentrations to exert proconvulsant effects, so the physiological relevance is unclear. However, it is known that the seizure facilitating effects of these steroids can be blocked by coadministration of allopregnanolone or other neurosteroids that positively modulate GABA_A receptors.⁷¹

In addition to anticonvulsant activity, there is some limited evidence that endogenous neurosteroids play a role in regulating epileptogenesis.^72–74 Following pilocarpine-induced status epilepticus in the rat, the neurosteroidogenic enzyme P450scc is upregulated for several weeks, suggesting that neurosteroidogenesis may be increased. Ordinarily rats develop spontaneous recurrent seizures following a latent period of similar duration to the period during which P450scc is elevated. Inhibiting neurosteroid synthesis with finasteride, accelerated the onset of spontaneous recurrent seizures, suggesting that endogenous neurosteroids play a role in restraining epileptogenesis or at least that they inhibit the expression of seizures. Exogenous treatment with neurosteroids or with progesterone, which serves as a precursor for neurosteroid synthesis, has also been reported to delay the occurrence of epileptogenesis in some situations.⁷⁵ In fact, progesterone may impair epileptogenesis in kindling models, even at doses that do not affect seizure expression.^76,77 If endogenous neurosteroids can be confirmed as endogenous regulators of epileptogenesis, neurosteroids themselves or modulators of neurosteroid disposition could potentially have disease-modifying therapeutic activity.

ROLE OF ENDOGENOUS NEUROSTEROIDS IN THE MODULATION OF SEIZURES

Endogenous neurosteroids may play a role in the physiological regulation of seizure susceptibility in individuals with epilepsy. We will discuss several such situations: catamenial epilepsy, stress, temporal lobe epilepsy, and alcohol withdrawal. However, it is noteworthy that there is no evidence that alterations in neurosteroid levels in the absence of preexisting epilepsy can induce epileptogenesis.

GANAXOLONE AS A NOVEL NEUROSTEROID-BASED ANTIEPILEPTIC DRUG

Ganaxolone, the synthetic 3β-methyl derivative of allopregnanolone,¹¹⁸ is the only neurosteroid that has been evaluated for the treatment of epilepsy in humans.^75,119 Allopregnanolne itself has been administered to humans at low doses intravenously (0.05–0.09 mg/kg) and found to be largely free of side effects except for sedation.^120,121 However, it has been proposed that allopregnanolone can undergo back conversion by 3α-HSOR isoenzymes to a hormonally active intermediate (dihydroprogesterone).¹²² The 3β-methyl substituent of ganaxolone eliminates this back conversion, potentially avoiding hormonal side effects. Other than this theoretical advantage, ganaxolone has pharmacological properties similar to the natural neurosteroid from which it was derived.

Preclinical studies

Ganaxolone has protective activity in diverse rodent seizure models, including clonic seizures induced by the chemoconvulsants pentylenetetrazol, bicuculline, flurothyl, t-butylbicycloorthobenzoate, aminophylline; limbic seizures in the 6 Hz model; amygdala and cocaine-kindled seizures; and corneal kindled seizures (Table 1).^{62,67,123–126} In chronically treated rats, tolerance does not occur to the anticonvulsant activity of ganaxolone.⁶⁷ In addition, a recent study in female amygdala kindled mice demonstrated suppression of behavioral and electrographic seizures with ED₅₀ of 6.6 mg/kg.¹²⁷

Table 1

Anticonvulsant Profile of Ganaxolone in Mouse Seizure Models.

Animal pharmacokinetic studies have found that ganaxolone has a large steady-state volume of distribution (6.5, 7.0, 19.5 and 3.5 L/kg in mice, rats, rabbits and dogs, respectively) indicating that it distributes extensively into tissues.⁷⁵ Studies with radioactive ganaxolone in rats have found that ganaxolone (and its metabolites) are concentrated in tissues including the brain (brain-to-plasma concentration ratio between 5 and 10). Ganaxalone is highly bound to human plasma proteins (>99%). It is extensively metabolized to at least 16 different compounds; the primary metabolite is 16α-hydroxyganaxolone, which likely results from the action of CYP3A4. This primary metabolite is inactive in the PTZ seizure model and is 25-fold weaker than ganaxolone in inhibiting [³⁵S]TBPS binding. Ganaxolone is a CYP3A4 autoinducer in rodents but not dogs or humans; chronic exposure to high doses in female rats does cause liver hypertrophy. Metabolites of ganaxolone are eliminated in the urine (13–23%) and feces (65–76%) in rats and dogs; the corresponding values in male healthy volunteers is 25% and 69%. Because of its aqueous insolubility, orally administered ganaxolone is poorly absorbed. To provide more consistent bioavailability, the steroid has been administered as a submicron particulate suspension and in a proprietary solid formulation.

Animal safety studies have demonstrated little evidence of target organ or systemic toxicity with either single-dose or multiple-dose ganaxolone treatment. In studies on pre- and postnatal development in mice, rats and dogs, ganaxolone did not affect fetal implantation, viability, or growth and development from birth to weaning, and was not teratogenic. Genotoxicity tests have not demonstrated any mutagenic or clastogenic potential for ganaxolone. Oral administration of ganaxolone to conscious dogs at a dose of 10 mg/kg did not reveal changes in cardiovascular hemodynamics.

CONCLUSIONS

Neurosteroids are endogenous modulators of neural excitability that are believed to have a role in the regulation of seizure susceptibility in the setting of preexisting epilepsy. Menstrual and stress related fluctuations in seizures may in part be related to changes in brain neurosteroid levels. In addition, men with TLE who have suppression of the hypothalamic-pituitary-gonadal axis may have a reduction in testosterone-derived neurosteroids that could worsen seizures.

Treatment with exogenously administered natural neurosteroids or synthetic analogs such as ganaxolone may be beneficial to treat partial seizures. Further studies are required to determine if neurosteroid replacement is a useful therapeutic approach for seizure exacerbations related to endogenous neurosteroid fluctutations, such as in catamenial epilepsy and stress. In the future, agents that influence the endogenous synthesis of neurosteroids, such as TSPO ligands, may find utility as an alternative to neurosteroids themselves in the treatment of epilepsy.^24,132

ACKNOWLEDGMENTS AND DISCLOSURES

The original research described in this article was supported in part by the NIH grants NS051398 and NS052158 (to D.S.R.) and NIH intramural grants NS002877 and NS002732 (to M.A.R.) and the Epilepsy Therapy Project (to M.A.R.). D.S.R. declares no conflicts; M.A.R. is a consultant to Sage Therapeutics and a scientific founder and has served as consultant to Marinus Pharmaceuticals, the current sponsor of ganaxolone.

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