Previous studies of the AZD0503/Saracatinib drug, a well-known fyn kinase inhibitor, have demonstrated excellent tolerability and bioavailability in humans. ISU researchers have discovered that the drug reduces seizure onset in the mouse kainate model of temporal lobe epilepsy. Thus, the drug has potential for the treatment of epilepsy in humans.
Chemoconvulsant-induced status epilepticus in rodents can lead to the development of temporal lobe epilepsy (TLE). ISU researchers unexpectedly found that the fyn kinase, a non-receptor tyrosine kinase, is up-regulated in microglial cells in a mouse kainate (KA) model of TLE. The researchers hypothesized that reducing the levels of fyn kinase would prevent seizure onset, decrease the severity of SE, and the brain pathology associated with epileptogenesis.
The fyn kinase hypothesis was tested in the fyn knockout mice (fyn -/-) with appropriate fyn+ /+ controls with saracatinib (25 mg/kg, oral, single dose) 4h prior to the induction of SE with KA (5mg/kg, i.p. at 30 min intervals or as a single high dose of 25 mg/kg). Two hours after SE establishment, the behavioral SE was terminated, the animals euthanized, and the brain, serum, and cerebrospinal fluid were collected for various analyses. Brain tissues were processed for Immunohistochemistry and western blot analysis. The time-dependent activation of fyn in microglial cells and initiation of neuro-inflammatory and neurodegenerative mechanisms in the hippocampus at 4h, 24h and 7day time points post-SE were also examined, and a electroencephalographic (EEG) analysis to determine the spike rate and SRS frequency was performed.
The behavioral studies revealed a significant reduction in the severity of the seizures and the time spent in convulsive motor seizures (CMS) stages in the fyn-/- mice and saracatinib treated mice during the 2h established SE. The mortality rate in the fyn-/- mice was lower than the fyn+/+ mice. The number of spontaneous electrographic non-convulsive seizures, the duration of CMS, and the spike frequency were also reduced in the fyn-/- mice. The epileptiform spike rate was higher in fyn+/+ mice during the first 7 days, but they decreased thereafter. IHC analysis of brain sections revealed a significant increase in fyn, PKC-δ, oxidative and nitrative stress markers (GP-91phox, 4-HNE, 3NT) in the microglia in the dentate gyrus, CA3 and CA1 at 4h and 24h post-SE in fyn+/+ mice. These levels were significantly reduced in the fyn-/- mice. Also, a significant increase in the fyn and PKC-δ nuclear translocation in reactive microglia at 24h post SE was detected in the fyn+/+ mice vs. the fyn-/- mice, suggesting their role in the pro-inflammatory responses. Quantitative RT-PCR analysis revealed an increase in the TNF-α, IL-1β and iNOS mRNA levels in controls when compared with the fyn-/-mice. In the 24h group, hippocampal western blots revealed down-regulation of fyn, PKC-δ, phospho Src-416, phospho PKCδ-507, GP-91phox, 4-HNE and caspase-3 levels in fyn-/-mice vs. fyn+/+mice. Moreover, an increase in the numbers of FJB-positive neurons was observed in the CA3 and CA1 regions of the hippocampus at 24h post-SE in fyn+/+ mice vs. fyn-/- mice.