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-D-aspartate (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 3alpha-ol-5beta-pregnan-20-one hemisuccinate (3alpha5betaHS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3alpha5betaHS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3alpha5betaHS 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 3alpha5betaHS 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.

Figure 1

Chemical structure of 3α5βHS. Sulfate replaces succinate at position 3 on the steriod A ring in 3α5βS.

Figure 2

3α5βHS rapidly inhibits the NMDA-induced whole cell currents. V_H = −70 mV. 3α5βHS (100 μM) inhibits the current induced by 30 μM NMDA in rat cortical neurons (A), rat hippocampal neurons (B), and chicken spinal cord neurons (C). The horizontal bar above each trace represents the period of drug application.

Figure 3

3α5βHS protects neurons against acute NMDA-induced cell death by decreasing the potency and efficacy of NMDA as an excitotoxin. (A) Dose–response curves for NMDA-induced neuronal death were determined using 15-min exposure to NMDA. 3α5βHS or DMSO vehicle was present during NMDA exposure only. 3α5βHS (100 μM) increases the EC₅₀ for NMDA-induced neuronal death from 31 ± 2 μM to 78 ± 28 μM (P < 0.05) and decreases the maximum neuronal death from 80 ± 2% to 68 ± 3% (P < 0.05); vehicle, n = 16; 3α5βHS, n = 5. (B) Dose–response curves for steroid modulation of NMDA-induced neuronal death were determined using 15-min exposure to 30 μM NMDA. 3α5βHS was present during NMDA exposure only. 3α5βHS inhibits NMDA-induced neuronal death with an EC₅₀ of 44 ± 14 μM and a maximum inhibition of 78 ± 12% (n = 4). Results are expressed as mean % neuronal death-control ± SEM. Smooth curves were generated by nonlinear regression using the logistic equation. The break in the x axis represents a change from linear to log scale.

Figure 4

3α5βHS inhibits NMDA-induced seizures and inhibits the late phase of formalin-induced pain. (A) Mice were injected i.v. with steroid or vehicle control (n = 8) followed 2 min later by injection with NMDA (200 mg/kg, i.p.), and the latency to seizure was recorded. Animals were observed for a maximum of 30 min. 3α5βHS dose dependently increased the latency to NMDA-induced seizures. 3α5βHS at 25 mg/kg completely prevented seizures during the period of observation. 3α5βS (25 mg/kg) had no effect on the latency to seizure. 3α5βS (50 mg/kg) was required to increase the latency to convulse comparable to that seen with 12.5 mg/kg 3α5βHS. (B) Mice were injected i.p. with 3α5βHS (15 mg/kg i.p.) or vehicle control 5 min prior to injection of 20 μl of 1% formalin into the hind paw. The time spent licking the injected paw was monitored for 25 min at 5-min intervals. The early phase (0–5 min) was unaffected by administration of 3α5βHS, whereas the time spend licking the injected paw in the late phase (10–25 min) was significantly reduced from 177 ± 64 s to 66 ± 13 s (∗, P < 0.05, n = 8) when animals were treated with 3α5βHS.

Figure 5

3α5βHS is neuroprotective in an in vivo model of stroke. Rats were infused with vehicle or 6.9 mg/kg per h (6.9 mg/kg loading dose) of 3α5βHS, beginning immediately or 30 min after initiation of ischemia. Infusion of 3α5βHS was continued for an additional 22 h, at which time the rats were killed and their brains were stained with 2,3,5-triphenyl tetrazolium Cl. (A) Representative sections from animals receiving 3α5βHS and vehicle infusions immediately after initiation of ischemia. Infarct area appears pale. (B) When 3α5βHS was administered beginning immediately after the onset of ischemia the volume of cortical infarct was reduced from 206 ± 22 mm³ to 110 ± 21 mm³ (P < 0.005; vehicle, n = 9; 3α5βHS, n = 10). The subcortical infarct was reduced from 103 ± 6 mm³ to 76 ± 6 mm³ (∗, P < 0.005). (C) When 3α5βHS was administered 30 min after the onset of ischemia the volume of cortical infarct was reduced from 173 ± 15 mm³ to 106 ± 15 mm³ (∗, P < 0.005; n = 13), with no reduction apparent in the subcortical region.