A Novel Local FK506 Delivery System Results in Excellent Axons Regeneration

Tajdaran, Kasra 1, 2 ; Chan, Katelyn 1, 2 ; Shoichet, Molly S. 1, 2 ; Gordon, Tessa 2 ; Borschel, Gregory H. 1, 2

1. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2. Division of Plastic and Reconstructive Surgery, The Hospital for Sick Children

Purpose: Administration of FK506, an FDA approved immunosuppressant, has shown to enhance nerve regeneration following peripheral nerve injuries. However, the severe side effects of the systemically delivered FK506 has prevented clinicians from using this drug routinely. Therefore, we have developed a novel fibrin gel based local delivery system for FK506. In this study, for the first time, we analyzed the effectiveness of the FK506 local delivery system to promote axon regeneration following peripheral nerve injury. In addition, we analyzed FK506 transport to the surrounding tissues, in vivo, from the delivery system at the nerve injury site.

Methods: FK506 was incorporated into fibrin gel in solubilized, particulated or poly(lactic-co-glycolic) acid microspheres-encapsulated forms. To analyze the effectiveness of the delivery systems in enhancing nerve regeneration, a rat nerve transection model was used, where the proximal tibial nerve stump was cross-sutured to the distal stump of a cut common peroneal nerve. Rats in the negative control groups either did not receive any delivery system treatment or received fibrin gel with empty microspheres. The experimental groups included rats treated with fibrin gel loaded with the three forms of FK506 formulation. Three weeks after repair, nerve regeneration was assessed using retrograde labeling and collecting nerve samples 7 mm distal to the repair site for histomorphometric analysis. Using mass spectrometry, FK506 tissue concentrations were analyzed at the site of the nerve injury, in sciatic nerve, dorsal root ganglia (DRGs), spinal cord, brain, heart, liver, kidney, and plasma at 7, 14, and 28 days post repair.

Results: Rats in experimental groups receiving FK506-loaded microspheres and the particulated FK506 had significantly highest number of motor and sensory neurons that regenerated their axons and allowing all tibial motoneurons regeneration. Histomorphometric analysis indicated increased number of myelinated axons following particulated FK506 and FK506 microspheres treatment compared to the negative control groups. The duration of FK506 in vivo release at the site of nerve injury was the shortest in the solubilized form for 7-days, then the particulate form for 14-days. The most prolonged release period was seen with the PLGA microsphere-encapsulated form for 28-days. The highest FK506 tissue concentration was detected within the entire spinal cord at day 7 regardless of the delivery system formulation. FK506, in vivo, was also found at the nerve injury site, sciatic nerve, DRGs, and the surrounding gluteal muscles, decreasing in concentration over time, with little to no drug detection in other vital organs.

Conclusion: The local application of FK506 via our proposed delivery systems resulted in excellent axon regeneration while minimizing the toxicity of systemic FK506 that has prevented clinicians from using FK506 routinely for treating severe cases of peripheral nerve injuries.