Investigating the efficacy and mechanistic pathways of saracatinib, a medical countermeasure, in a pediatric rat DFP model - Abstract The organophosphate nerve agent (OPNA) attacks in Tokyo, Syria, Malaysia, and England prove the real threat of OPNAs. Acute exposure to OPNA impacts human health globally, and we lack treatment for survivors. Until recently, preventing acute death due to OPNA exposure was a priority. However, addressing the long- term effects is also critical given that survivors, though hospitalized and treated with conventional therapy, developed seizures and cognitive, motor, and psychological impairments. OPNAs are cholinesterase inhibitors and potent seizurogenics. In animal models, acute OPNA exposure induces status epilepticus (SE) and other cholinergic symptoms. The current medical countermeasures (MCM) do not prevent long-term neurotoxicity and comorbidity, which are primarily due to persistent neuroinflammation and neurodegeneration. Our overarching hypothesis is that a neuroprotectant, in combination with MCM, will effectively counteract OPNA- induced long-term neurotoxicity and restore brain function. In the currently funded U01 project, we optimized the dose of two novel neuroprotectants, saracatinib (SAR/AZD0530, a Src kinase inhibitor) and 1400W (an inducible NO synthase inhibitor) and demonstrated their long-term disease-modifying effects in adult rat DFP and soman models. We have also shown the feasibility and pharmacokinetics of diet-incorporated SAR in long term studies in adult rat DFP and soman models. Based on the U01 project outcomes, we are preparing for a pre-IND meeting with the FDA to develop SAR for clinical trials. AstraZeneca (AZ), who discovered SAR, has supplied the test drug for all our preclinical studies through Open Innovation. AZ also supports the pre- IND process by providing critical preclinical and clinical data they have acquired from various clinical studies for many years. However, the significant gap in this process is the lack of pediatric data for SAR. In this Administrative Supplement project, we will test the long-term efficacy and tolerability of SAR in the diet in a pediatric rat DFP model (postnatal day 21) in both sexes as adjunct therapy. We will treat control littermates with a control diet (without SAR). We will maintain the animals on SAR in-diet throughout their adulthood, perform behavioral tests, and euthanize at 2 months post-DFP and some animals at one-week post-DFP. We will do multiplex cytokine/chemokine assays from serum and CSF as readouts for the effects of SAR and correlate with other outcomes. The brain tissues will be used for phosphoproteomics to identify differentially phosphorylated proteins and correlate these with behavioral outcomes. Based on the hits, we will validate the top 6-8 proteins of relevant pathways using the Westerns and confirm their cellular localization in brain sections for comprehensive molecular and cellular insights. Enrichment of specific subsets of the phosphoproteomes such as phosphotyrosine and phospho-motif enrichment of kinase substrates will reveal targets of SAR and DFP. This research is within the CCRP scope of current U01 (NS117284) and addresses NOT-AI-24-022, i.e., to promote the development of pediatric chemical research models and MCM discovery.
