Sex-specific hypnotic effects of the neuroactive steroid (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile are mediated by peripheral metabolism into an active hypnotic steroid

Background: The novel synthetic neuroactive steroid (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH) blocks T-type calcium channels but does not directly modulate neuronal γ-aminobutyric acid type A (GABA_A) currents like other anaesthetic neurosteroids. As 3β-OH has sex-specific hypnotic effects in adult rats, we studied the mechanism contributing to sex differences in its effects. Methods: We used a combination of behavioural loss of righting reflex, neuroendocrine, pharmacokinetic, in vitro patch-clamp electrophysiology, and in vivo electrophysiological approaches in wild-type mice and in genetic knockouts of the Ca_V3.1 T-type calcium channel isoform to study the mechanisms by which 3β-OH and its metabolite produces sex-specific hypnotic effects. Results: Adult male mice were less sensitive to the hypnotic effects of 3β-OH compared with female mice, and these differences appeared during development. Adult males had higher 3β-OH brain concentrations despite being less sensitive to its hypnotic effects. Females metabolised 3β-OH into the active GABA_A receptor positive allosteric modulator (3α,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3α-OH) to a greater extent than males. The 3α-OH metabolite has T-channel blocking properties with sex-specific hypnotic and pharmacokinetic effects. Sex-dependent suppression of the cortical electroencephalogram is more pronounced with 3α-OH compared with 3β-OH. Conclusions: The sex-specific differences in the hypnotic effect of 3β-OH in mice are attributable to differences in its peripheral metabolism into the more potent hypnotic metabolite 3α-OH.