N3: Negative, Nerve-Targeted Nanocarriers

To defeat OP nerve agents, we must match both their potency and access to the synaptic cleft. Unfortunately, the most potent organophosphate antidotes cannot access neurons from the systemic circulation. Phosphotriesterase (PTE) enzyme mutants are too large, and small, cationic oximes are too charged to cross the blood-brain-barrier or blood-nerve-barrier in appreciable quantities. State-of-the-art PTE therapy requires weekly prophylactic use as a blood-based scavenger; immunogenicity concerns and costly recurring doses dim its prospects for success in the field. Similarly, standard medicinal chemistry techniques for enhancing oxime access to nerves (e.g. lipophilization, prodrug design, hijacking active transporters) have failed to bear fruit. In this innovative Phase II effort, nSI will adapt our Phase I formulation and analytical workflows to handle (a) in vivo fluid volumes, (b) the formulation purification required for pharmacokinetic / biodistribution (PK/BD) trials in vivo, and (c) the use of high-activity PTE mutants instead of a PTE simulant. Using this scaled workflow, we will substitute PTE mutants for PTE simulants and assess impact on formulation parameters (and enzyme activity) for each formulation in our library, and expand our library from 50 to >200 passing formulations. Furthermore, we will continue work on formulation barcoding that began in Phase I, demonstrating (a) that barcode attachment does not impact formulation properties or PTE activity, and (b) successful formulation multiplexing capability in vitro. Finally, for each formulation in the library, we will also conduct in vitro stability studies (in plasma and at 37oC) and in vitro cytotoxicity and nanoparticle (NP) association/uptake studies using primary human neurons. The result will be a large, multiplexed, multidrug NP library that can be co-administered and rapidly screened for in vivo neurotropism in Phase III. If successful, the resulting flexible neurotropic delivery platform could solve critically important medical problems in pain management, psychiatric and CNS disorders, and countering deadly nerve agents.