Degradable Polymer Constructs Delivering AG1478 to Promote Axon Regeneration in the Mature Mammalian Central Nervous System

Neurons fail to regenerate after injury due to inhibition at the intrinsic and extrinsic level. Promoting nerve regeneration involves not only modulating the post-injury microenvironment but also ensuring survival of injured neurons. There is evidence that sustained administration of reversible or irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) promotes significant nerve regeneration and increases neuronal survival after injury. However, means of efficient delivery of TKIs have been limited. A platform that can deliver EGFR TKIs in an efficient, sustained, and minimally invasive manner is needed. We developed an injectable sustained-released platform using the degradable polymer poly(lactic-co-glycolic acid) (PLGA). The reversible EGFR TKI 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) was encapsulated in PLGA to make microspheres and nanospheres using a single-emulsion solvent evaporation technique. We tested three different formulations to increase encapsulation and reduce sphere size: solid-in-oil-in-water, oil-in-water, and oil-in-water with co-solvent. Addition of the co-solvent trifluoroethanol increased loading of AG1478 by 125% compared to solid-in-oil-in-water and 38% compared to oil-in-water, and decreased microsphere diameter significantly (P < 0.001). Co-solvent addition also prolonged AG1478 release to over six months. Once released, AG1478 remained bioactive and inhibited EGFR in immortalized rat fibroblasts and EGFR-amplified human carcinoma cells. We evaluated the effectiveness of this system in promoting nerve regeneration using a rodent optic nerve crush injury model. We encapsulated coumarin-6 in PLGA spheres for tracking purposes and at two weeks we detected spheres in the vitreous, but at four weeks only nanospheres could be detected. The persistence of AG1478 in the vitreous was different for microspheres and nanospheres. We recovered drug from the vitreous of nanosphere-injected animals, but not microsphere-injected animals, after two weeks. AG1478 microspheres and nanospheres promoted optic nerve regeneration at two weeks, but at four weeks evidence of regeneration was found only in the nanosphere-injected animals. These data demonstrate the potential of these constructs as an injectable platform for EGFR TKI administration and the promise of this technology to promote nerve regeneration after acute central nervous system injury.