Development of a small-molecule epigenetic regenerative therapy. Subcutaneous administration of alginate formulations with high loads of zebularine and retinoic acid promotes tissue growth, vascularization and innervation and induces extensive epigenetic repatterning

Recently, zebularine, a small-molecule epigenetic inhibitor and retinoic acid, acting as a transcriptional activator, have been found to induce tissue regeneration. In this study, the pro-regenerative properties of zebularine and retinoic acid were combined with the potential of the alginate carrier to expand its therapeutic possibilities. Alginate formulations of zebularine and retinoic acid were developed for subcutaneous administration to mice. Hydrophilic zebularine formed a homogenous formulation with extreme drug loadings reaching 240 mg of zebularine per 1 ml of 2% sodium alginate, while hydrophobic retinoic acid, 0.8 mg/ml, dispersed as fine crystals. Cell culture tests exhibited no significant cytotoxicity of the alginate formulations. Subcutaneous administration of zebularine and retinoic acid in 2% sodium alginate promoted regenerative responses in a mouse model of ear pinna punch wound mice involving the restoration of tissue architecture, nerve and vessel growth, and extensive epigenetic and transcriptional repatterning with no adverse effects observed in the animals. Significant trancriptomic responses to the epigenetic treatment included the induction of epithelium development genes contrasted with the downregulation of muscle development genes on day 7 post-injury. Among the remarkable changes in global gene methylation are those in neurodevelopmental genes. In vitro studies showed rapid zebularine but no retinoic acid discharge from the alginate formulations. Live ultrasound imaging demonstrated gradual absorption of the subcutaneously injected alginate formulations, which may explain the in vivo activity of retinoic acid following subcutaneous administration. Effective induction of tissue regeneration together with a high safety profile and of the subcutaneously administered pro-regenerative alginate formulations opens the way to testing further regenerative therapies for hard-to-reach lesions.