| Conantokin-G (Con-G or CGX-1007), a potent NR2B subunit selective NMDA receptor antagonist, was evaluated for its neuroprotective properties in experimental models of neuronal injury. In primary neuronal cultures Con-G was shown to decrease excitotoxic calcium responses to NMDA and provide 100% neuroprotection against hypoxia/hypoglycemia (34 μM[13–91]), NMDA (77 μM[42–141]), glutamate (819 μM[346–1937]) or veratradine (2136 μM[1508–3026]) induced injury (numbers in parentheses indicate EC50 and 95% confidence limits). Con-G (0.1–1 μM) also provided up to 80% protection against staurosporine-induced apoptotic injury (P < 0.01, n = 12/group), which was linked to the NR2B subunit. For in vivo brain injury studies, middle cerebral artery occlusion (MCAo) in the rat was used as a model of transient focal brain ischemia. In this model Con-G (0.01–2.0 nmoles, i.c.v.) reduced brain infarction and improved both neurological and electroencephalographic (EEG) recovery as evaluated both 24 and 72 h post-injury. The maximal neuroprotective effect was measured with the highest dose of Con-G tested (2.0 nmol, i.c.v) with an 89% reduction of core infarct volume (P < 0.05, n = 6–10/group). Post-injury time course experiments demonstrated a therapeutic window out to at least 4 h from the start of the injury. These neuroprotective effects were also associated with a 50% reduction in the early expression (i.e. 1–4 h) of the c-fos gene (P < 0.05, n = 3–4/group), a preservation of Bcl-2 immunoreactivity at 24 h (P < 0.05, n = 4), and with a reduction in DNA strand breaks in the ischemic hemisphere as evaluated 24 h post-injury (P < 0.05, n = 6/group). Clinically relevant routes of administration were evaluated by administering intrathecal (i.t.) injections of Con-G (20–160 nmol), which provided up to 62% reduction in brain infarction (P < 0.05, n = 8–9/group) along with significant neurlogical recovery and a therapeutic window of up to 8 h post-injury. Con-G (i.t.) treatment was also associated with fewer ischemia-induced EEG seizures with a positive albeit non-significant trend (P > 0.05, n = 6–7/group) between the number of brain seizures and infarct volume. Intrathecal Con-G was not associated with changes in c-fos gene expression although, similar to i.c.v. administration, Bcl-2 immunoreactivity was preserved in cortical tissues (P < 0.05, n = 3/group) and presence of TUNEL positive cells decreased at 24 h (P < 0.05, n = 6/group). These data provide evidence for the potent and highly efficacious effect of Con-G as a neuroprotective agent with an excellent therapeutic window for the potential intervention against ischemic/excitotoxic brain injury in humans. |
