Neuroprotective activity of a new class of steroidal inhibitors of the N -methyl- d -aspartate receptor

Abstract

Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-Daspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3␣-ol-5␤-pregnan-20-one hemisuccinate (3␣5␤HS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3␣5␤HS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3␣5␤HS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3␣5␤HS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.Exposure of neurons to the excitatory neurotransmitter glutamate causes an increase in the concentration of intracellular free Ca 2ϩ (1) and initiates the process of excitotoxic cell death (2). There are at least three pharmacologically distinct ionotropic glutamate receptors that differ in their sensitivity to the selective agonists N-methyl-D-aspartate (NMDA), ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate. Whereas excessive activation of NMDA, AMPA, or kainate receptors results in neurotoxicity, specific inhibition of NMDA receptors is sufficient to attenuate most of the neuronal death that develops after in vitro hypoxia, exposure to glutamate, in vivo ischemia, or hypoglycemia (3). As Ca 2ϩ passes through the NMDA receptor operated ion channel, it is believed that this receptor subserves a crucial role in mediating excitotoxicity. Therefore, it has been proposed that over activation of NMDA receptors may be an obligatory phase preceding neuronal death that occurs following focal cerebral ischemia induced stroke in humans.Neuroactive steroids have been shown to directly modulate excitatory and inhibitory amino acid receptor function (4-7). Therefore the possibility is raised that certain steroids might be useful neuroprotective agents. The endogenous neurosteroid pregnenolone sulfate probably acts as a positive allosteric modulator of the NMDA receptor by enhancing NMDAinduced inward currents and the subsequent rise in cytoplasmic free calcium (8, 9), whereas 3␣-ol-5␤-pregnan-20-one sulfate (3␣5␤S) is a negative modulator of NMDA-induced currents (6) and inhibits NMDA-stimulated increases in intracellular calcium (10). The present study examines the in vitro and in vivo neuroprotective activity of 3␣-ol-5␤-pregnan-20-one hemisuccinate (3␣5␤HS) ( Fig. 1; a synthetic analog of the endogenou...

Molecular Mechanisms for Nanomolar Concentrations of Neurosteroids at NR1/NR2B Receptors

“…Neurosteroids, and sulfated neurosteroids in particular, are present at relatively low concentrations in the rat brain (Higashi et al, 2003;Liu et al, 2003). Nonetheless, when PS is administered at femtomole concentrations by intracerebroventricular injection to rodents, processes involving the NMDA receptor are affected (Flood et al, 1995;Mathis et al, 1996;Meziane et al, 1996;Weaver et al, 1997). In contrast, in electrophysiological experiments, much higher (micromolar) concentrations of the steroids are required to achieve modulatory effects on recombinant NMDA receptors (Mukai et al, 2000;Malayev et al, 2002).…”

“…The interaction between PS (at nanomolar concentrations) and ifenprodil as seen in our experiments may thus involve other site(s) of action for PS compared with those studied at much higher concentrations. These targets can represent high-and low-affinity sites for some neurosteroids where the former sites may mediate in vivo effects seen after administration of very low amounts of these compounds (Flood et al, 1995;Mathis et al, 1996;Meziane et al, 1996;Weaver et al, 1997). We propose that PS and 3␣5␤S at physiological concentration can change ifenprodil binding by converting the receptor site from a one-site state into a twosite state.…”

Targeting the Modulation of Neural Circuitry for the Treatment of Anxiety Disorders

“…Synthetic analogs of neuroactive steroids, which are more resistant to metabolism and better able to cross the blood-brain barrier, are now being investigated for potential use as anxiolytics, anesthetics, and anticonvulsants (Weaver et al, 1997;Gasior et al, 1999). One synthetic neurosteroid, pregnanolone hemisuccinate, produces sedation in mice and rats (Weaver et al, 1997), raising the possibility that it acts either by inhibiting the NMDA receptor or by crossing the bloodbrain barrier and undergoing metabolism to pregnanolone. Synthetic neurosteroids bearing a hemisuccinate group are more resistant to hydrolysis than the corresponding sulfate esters and are partly unionized at physiologic pH, allowing ready passage across the blood-brain barrier.…”

“…Neurosteroids such as pregnanolone sulfate, which enhances cognition, positively modulates NMDAR function, and stimulates NMDAR trafficking to the cell surface (Wu et al, 1991;Kostakis et al, 2013), may represent one such endogenous mechanism for linking fearful stimuli and the emotional response subject to therapeutic manipulation (Plescia et al, 2013). Neuroactive steroids, such as pregnanolone sulfate and pregnanolone hemisuccinate, which inhibit glutamatergic neurotransmission by negatively modulating NMDAR function (Park-Chung et al, 1994) and may also exhibit mixed function by serving as both a negative modulator of the NMDAR and a prodrug for pregnanolone (a positive modulator of GABA A R) delivery across the blood-brain barrier, may also represent a novel platform for discovery (Weaver et al, 1997(Weaver et al, , 2000.…”

Brain Plasticity Following Ischemia: Effect of Estrogen and Other Cerebroprotective Drugs

“…Estrogen increases the number of surviving cells following ischemia (Liu et al, 2009;Merchenthaler et al, 2003;Platha et al, 2004;Wappler et al, 2010), reduces excitotoxicity (Connell et el., 2007;Herson et al, 2009;Weaver et al, 1997), inflammation (Herson et al, 2009 ;Stein, 2001;Suzuki et al, 2007), moderates blood-brain barrier dysfunction (Liu et al, 2005), is antioxidant (Connell et el., 2007), increases cerebral blood flow (Herson et al, 2009;Hurn et al, 1995;Pelligrino et al, 1998), reduces spontaneous postischemic hyperthermia (Platha et al, 2004), etc. Cerebral ischemia studies in ER-and ER-KO mice models, and pharmacological receptor inhibition have shown that ER-is the primary mediator of neuroprotection.…”