Allopregnanolone Analogs That Positively Modulate GABA_A Receptors Protect against Partial Seizures Induced by 6‐Hz Electrical Stimulation in Mice

Abstract

Summary:Purpose: Low-frequency (6 Hz), long-duration (3 s) electrical stimulation in mice produces seizures characterized by immobility, focal clonus, and automatic behaviors reminiscent of human limbic epilepsy. Renewed interest has been expressed in this seizure model with the recognition that it is sensitive to a broad spectrum of anticonvulsants (AEDs) and may have distinct pharmacologic responsiveness from other in vivo tests of AED efficacy. Here we sought to determine whether the progesteronederived neuroactive steroid allopregnanolone (5α,3α-P) and several structural analogues with varying degrees of activity as positive allosteric modulators of γ -aminobutyric acid (GABA) A receptors are protective in the 6-Hz seizure model.Methods: Mice were pretreated with neuroactive steroids (15 min before) or clonazepam (CZP; 30 min before) to 6-Hz corneal stimulation (32 mA). Animals that failed to exhibit immobility were considered protected. Results:The neuroactive steroids prevented 6-Hz seizures with rank order of potencies (ED 50 values): ganaxolone (6.3 mg/kg) > 5α,3α-P (14.2 mg/kg) ≥ 5β,3α-P (14.4 mg/kg) > 5α,3β-P (>100 mg/kg). CZP also was protective (ED 50 value, 0.075 mg/kg). The potencies of the neuroactive steroids and CZP are similar to their previously reported activities in the pentylenetetrazol (PTZ) seizure model.Conclusions: Neuroactive steroids have comparable potencies in the 6-Hz and PTZ models. Their structural specificity in both models corresponds with their activities as positive allosteric modulators of GABA A receptors, although ganaxolone is more potent than expected, probably because it has greater bioavailability. The 6-Hz model may be a valuable tool in drug development for the identification of GABAergic AEDs.

The Neuropharmacology of the Ketogenic Diet

“…The lack of activity in mice raises doubt that effects on GABA mechanisms contribute in a substantial way to the seizure protection conferred by the diet. Recently, we found that the ketogenic diet is highly protective against 6 Hz electroshock seizures in mice, a model of limbic seizures that shows a similar sensitivity to anticonvulsant agents that act on GABA systems as does the pentylenetetrazol test [32,33] (A.L.H., M. Lyle, M.G., and M.A.R., unpublished data). In particular, anticonvulsant agents that target GABA systems are highly effective and relatively potent in the 6 Hz model.…”

“…Reduced availability of oxaloacetate along with robust availability of α-ketoglutarate from high activity of the first part of the Krebs cycle leads to low aspartate levels. It has been hypothesized that more glutamate is thus accessible to glutamic acid decarboxylase for production of GABA [33]. Not all Krebs cycle intermediates are shown in the schematic.…”

Mass spectrometric assay and physiological–pharmacological activity of androgenic neurosteroids

“…However, 3α-androstanediol at normal ED 50 dosage does not protect seizures induced by gluatamate receptor agonists such as kainic acid, NMDA and 4-aminopyridine (Reddy, 2004b). 3α-Androstanediol is structurally similar to allopregnanolone and conferred seizure protection in the 6-Hz electroshock model of epilepsy (Kaminski et al, 2004). Overall, the anticonvulsant profile of 3α-androstanediol is highly consistent with other GABA A receptor modulating neurosteroids including allopregnanolone and THDOC, which have similar spectrum of anticonvulsant activity in animal seizure models (Belelli et al, 1989;Kokate et al, 1994;Reddy and Rogawski, 2002;Reddy et al, 2004;Reddy, 2006).…”

Ganaxolone

“…5 GNX prevented clonic and behavioral seizures caused by low-frequency (6 HZ) long-duration (3 s) electrical stimulation in mice. 6 PTZ-induced seizures in rats did not develop tolerance to chronic treatment with GNX. 7 …”