Background: Ischemic stroke is one of the leading causes of mortality and morbidity in the world and
effective neuroprotectants are yet to be developed. Recent studies have demonstrated excellent neuroprotective
effects of a bivalent enkephalin opioid agonist, biphalin in multiple stroke models.
Methods: The purpose of this study is to evaluate novel multifunctional enkephalin-fentanyl opioid agonists,
LYS436, LYS739 and LYS416 for their neuroprotective potential using in vitro and in vivo ischemic stroke
models and to compare the effect to that of biphalin.
Results: In general, all non-selective opioid agonists significantly decreased neuronal cell death and levels of
reactive oxygen species in primary neurons subjescted to hypoxia-aglycemia/re-oxygenation or NMDA neurotoxicity.
Fluorinated enkephalin-fentanyl conjugate, LYS739 showed enhanced neuroprotection in both in vitro
models compared to biphalin. Based on further in vitro screening and comparative studies to biphalin, LYS739
was selected as a lead for in vivo experimentation. A mouse middle cerebral artery occlusion (MCAO) stroke
model was utilized to study biphalin and the lead analog, LYS739. Both agonists significantly decreased brain
infarct and edema ratios compared to saline treated group. Neurological impairment after stroke was statistically
significantly improved in terms of neurological score and locomotor activities with LYS739 and biphalin treatment.
Importantly, LYS739 and biphalin demonstrated better neuroprotection compared to fentanyl, and this
effect was reversed by non-selective opioid antagonist naltrexone.
Conclusion: In summary, the results of this study suggest that the multifunctional fluorinated enkephalin analog,
LYS739 can be considered as a potential lead for ischemic stroke research and may provide advantages given the
multimeric peptide-opiate structure.