In vivo proof of concept:pH-dependent neuroprotection
DESCRIPTION (provided by applicant): Neurodegeneration accompanying stroke and certain types of seizure is a significant problem that has been refractory to treatment despite intensive investigation in both the public and private sector. Neuronal degeneration in animal models can be substantially reduced by glutamate receptor antagonists of the NMDA receptor subtype, suggesting that activation of this receptor exacerbates the neuronal death. Unfortunately, NMDA antagonists in clinical development have proved toxic at doses necessary to provide benefit. We have identified a strategy for overcoming these limitations, namely to identify compounds that are more effective NMDA receptor blockers at the acidic pH characteristic of ischemic or seizing brain tissue. We hypothesize that the pH drop during stroke or convulsive activity might be harnessed as a switch to activate neuroprotective agents. In this way side effects caused by receptor block in unaffected tissue could be minimized because drug at these sites would be less active. We envision developing a drug that is virtually inactive at normal brain pH but rapidly blocks NMDA receptors in stroked or seizing tissues as soon as the pH drops. We have designed a moderate throughput frog oocyte assay to screen drugs for pH-dependent block at the ifenprodil site of the NMDA receptor. Using this assay, we have identified novel pre-lead compounds that display submicromolar potency at pH 6.9. A key unresolved issue for evaluating our overall strategy is whether the pHdependence of NMDA receptor block in vitro is predictive of neuroprotective activity in vivo; that is, whether the pH swings during neuropathological events are large enough to activate NMDA receptor block. The purpose of this SBIR is to seek this proof of principle in animal models. The Phase I goals are:]. To test whether the pH drop in the ischemic penumbra can activate the neuroprotective effect of selected NMDA receptor antagonists. We will compare the neuroprotective effects of structurally related pairs of novel NMDA receptor blockers or structural isomers with similar potency at physiological pH but different potency at acidic pH. The neuroprotective effect of intracerebralventricular (ICV) injection of compounds into mice subjected to intraluminal occlusion of the middle cerebral artery will be assessed 24 hours post-ischemia. Active compounds also will be administered intraperitoneal (IP) to evaluate brain penetration. 2. To determine whether NMDA receptor blockers showing strong pH-dependence of block will reduce neuron injury and duration of seizures. Pairs of structurally-related compounds that are systemically active in the ischemia model will be injected IP into mice before inducing status epilepticus. The intensity and duration of seizure state will be assessed behaviorally, and the extent of neuronal injury will be measured three days later.
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