4 - Development of Neurosteroid-Based Novel Psychotropic Drugs

This chapter describes current knowledge of endogenous neurosteroids, namely, their mechanism of action and therapeutic benefits in epilepsy, anxiety, and other neurological conditions. Neurosteroids are synthesized de novo within the nervous system and have rapid effects on neuronal excitability. A variety of neurosteroids are present in the brain including allopregnanolone (brexanolone), pregnanolone, allotetrahydrodeoxycorticosterone, and androstanediol. Synthesized from cholesterol, neurosteroids interact with a wide variety of neurotransmitter receptors including GABA-A receptors, N-methyl-d-aspartate, and other neuronal receptors. Pregnane and androstane neurosteroids are positive allosteric modulators of GABA-A receptors and have therapeutic potential as anticonvulsant and anxiolytic agents. Neurosteroid sulfates are negative allosteric modulators at GABA-A receptors and can alter excitatory neurotransmission within the brain via glutamate receptors. Neurosteroids play a key role in regulation of seizure susceptibility, anxiety, and other neuropsychiatric conditions. Neurosteroid sulfates provide benefits such as attenuation of memory and psychiatric symptoms. Both sulfated steroids and neurosteroids play a key role in endogenous neuroprotective functions in the brain.

This chapter describes current knowledge of endogenous neurosteroids, namely, their mechanism of action and therapeutic benefits in epilepsy, anxiety, and other neurological conditions. Neurosteroids are synthesized de novo within the nervous system and have rapid effects on neuronal excitability. A variety of neurosteroids are present in the brain including allopregnanolone (brexanolone), pregnanolone, allotetrahydrodeoxycorticosterone, and androstanediol. Synthesized from cholesterol, neurosteroids interact with a wide variety of neurotransmitter receptors including GABA-A receptors, N-methyl-d-aspartate, and other neuronal receptors. Pregnane and androstane neurosteroids are positive allosteric modulators of GABA-A receptors and have therapeutic potential as anticonvulsant and anxiolytic agents. Neurosteroid sulfates are negative allosteric modulators at GABA-A receptors and can alter excitatory neurotransmission within the brain via glutamate receptors. Neurosteroids play a key role in regulation of seizure susceptibility, anxiety, and other neuropsychiatric conditions. Neurosteroid sulfates provide benefits such as attenuation of memory and psychiatric symptoms. Both sulfated steroids and neurosteroids play a key role in endogenous neuroprotective functions in the brain.

The target of the current study is to formulate letrozole loaded nanoemulsion (LET-NE) for the direct nose to brain delivery to reduce peripheral effects of letrozole (LET). LET-NE is compared against intraperitoneally administered free LET in kainic acid (KA) induced status epilepticus (SE) in mice. LET loaded nanoemulsion (LET-NE) was prepared by aqueous microtitration method using Triacetin, Tween 80 and PEG-400 as the oil phase, surfactant, and co-surfactant. Nanoemulsion was studied for droplet size, polydispersity index (PDI), zeta potential, percentage transmittance, drug content, surface morphology. TEM images of developed formulation demonstrated spherical droplets with a mean diameter of 95.59 ± 2.34 nm, PDI of 0.162 ± 0.012 and zeta potential of −7.12 ± 0.12 mV respectively. In in-vitro and ex-vivo drug release, LET-NE showed prolonged drug release profile as compared to suspension. SE was induced by KA (10 mg/kg, i.p.) in Swiss albino mice. Behavioral seizure monitoring, biochemical estimations, and histopathological examination were performed. The onset time of SE was significantly enhanced and % incidence of SE was reduced by intranasal administration of LET-NE as compared to KA and LET administered intraperitoneally. Biochemical estimations revealed that LET-NE effectively decreased levels of 17-β estradiol while the levels of 5α-Dihydrotestosterone (5α-DHT) and 3α-androstanediol (3α-Diol) were significantly increased in the hippocampus. In cresyl violet staining LET-NE showed better protection of the hippocampus from neurotoxicity induced by KA as compared to LET. Also, in gamma scintigraphy of mouse brain, intranasal administration of nanoemulsion exhibited the presence of high concentration of LET. The study demonstrates the anticonvulsant and neuroprotective effect of LET-NE probably by inhibition of aromatization of testosterone into 17-β estradiol, proconvulsant, and diverting the pathway into the synthesis of testosterone metabolites, 3α-Diol with known anticonvulsant and neuroprotective action. Brain targeting of LET-NE showed better anticonvulsant and neuroprotective action than LET.

Evidence shows neurosteroids play a key role in regulating epileptogenesis. Neurosteroids such as testosterone modulate seizure susceptibility through its transformation to metabolites which show proconvulsant and anticonvulsant effects, respectively. Reduction of testosterone by aromatase generates proconvulsant 17-β estradiol. Alternatively, testosterone is metabolized into 5α-dihydrotestosterone (5α-DHT) by 5α-reductase, which is then reduced by 3α-hydroxysteroid oxidoreductase enzyme (3α-HSOR) to form anticonvulsant metabolite 3α-androstanediol (3α-Diol), a potent GABA_A receptor modulating neurosteroid. The present study evaluated whether inhibition of aromatase inhibitor letrozole protects against seizures and neuronal degeneration induced by kainic acid (KA) (10 mg/kg, i.p.) in Swiss albino mice. Letrozole (1 mg/kg, i.p.) administered one hour prior to KA significantly increased the onset time of seizures and reduced the% incidence of seizures. Pretreatment with finasteride, a selective inhibitor of 5α-reductase and indomethacin, a selective inhibitor of 3α-hydroxysteroid oxidoreductase enzyme (3α-HSOR), reversed the protective effects of letrozole in KA-induced seizures in mice. Microscopic examination using cresyl violet staining revealed that letrozole did not modify KA-induced neurotoxicity in the CA1, CA3 and DG region of the hippocampus. Letrozole treatment resulted in the reduced levels of 17-β estradiol and elevated the levels of 5α-dihydrotestosterone (DHT) and 3α-Diol in the hippocampus. Finasteride and indomethacin attenuated letrozole-induced elevations of 5α-DHT and 3α-Diol. Our results indicate the potential anticonvulsant effects of letrozole against KA-induced seizures in mice that might be mediated by inhibiting aromatization of testosterone to 17β-estradiol, a proconvulsant hormone and by redirecting the synthesis to anticonvulsant metabolites, 5α-DHT and 3α-Diol. Acute aromatase inhibition, thus, might be used as an adjuvant in the treatment of status epilepticus and can be pursued further.

Neurosteroids like allopregnanolone (AP) are positive allosteric modulators of synaptic and extrasynaptic GABA-A receptors. AP and related neurosteroids exhibit a greater potency for δ-containing extrasynaptic receptors. The δGABA-A receptors, which are expressed extrasynaptically in the dentate gyrus and other regions, contribute to tonic inhibition, promoting network shunting as well as reducing seizure susceptibility. Levels of endogenous neurosteroids fluctuate with ovarian cycle. Natural and synthetic neurosteroids maximally potentiate tonic inhibition in the hippocampus and provide robust protection against a variety of limbic seizures and status epilepticus. Recently, a consensus neurosteroid pharmacophore model has been proposed at extrasynaptic δGABA-A receptors based on structure–activity relationship for functional activation of tonic currents and seizure protection. Aside from anticonvulsant actions, neurosteroids have been found to be powerful anxiolytic and anesthetic agents. Neurosteroids and Zn^2 + have preferential affinity for δ-containing receptors. Thus, Zn^2 + can prevent neurosteroid activation of extrasynaptic δGABA-A receptor-mediated tonic inhibition. Recently, we demonstrated that Zn^2 + selectively inhibits extrasynaptic δGABA-A receptors and thereby fully prevents AP activation of tonic inhibition and seizure protection. We confirmed that neurosteroids exhibit greater sensitivity at extrasynaptic δGABA-A receptors. Overall, extrasynaptic GABA-A receptors are primary mediators of tonic inhibition in the brain and play a key role in the pathophysiology of epilepsy and other neurological disorders.