Development of recombinant BMP-7 with excipients for inhibition of scar formation to apply eye drops.

Abstract

Purpose: The purpose of this study is to apply recombinant BMP-7 with excipients for eye drop as a therapeutic reagent for scar formation.

Methods: 1) Inflammatory inhibition effect of rBMP-7 with excipient: J774A.1 cells were split 6 well plates. 2 days after, the cells were stimulated with LPS (500 ng/ml) and rBMP-7 (200 ng/ml) with Hyaluronic acid (0.3 % w/v) or Cyclodextrin (0.5% w/v) was treated. After 24 hours, the supernatant was collected and analyzed with TNF-α ELISA. 2) Cytotoxicity of excipients on HaCaT cells: HaCaT cells were prepared (2.5 X 103 cells/well) in a 96 wells microplate in a final volume of 100 ul/ well medium and Hyaluronic acid or Cyclodextrin was treated in dependence of concentration. After 24 hours, the supernatant was mixed with EZ-Cytox reagent and incubated for 1 hour. Finally, the absorbance of the samples was measured using a microtiter plate reader at 450 nm. 3) Effect of rBMP-7 with excipients in corneal wound healing on a corneal alkali injury model: nine-week-old male C57BL/6 mice were anesthetized with ketamine and proparacane, sequentially. Corneal alkali burn was induced with 3 ul of 0.15 N NaOH for 30 seconds to the both eyes and the eyes were washed with saline for 30 seconds. After 3 days, rBMP-7 (1000 pg/2ul) or excipients mixed with rBMP-7 was dropped to the right eye and saline was applied to the left eye 4 times a day for 5 days. The eyes were evaluated weekly for 2 weeks and corneas were separated from fixed eye balls with 2% paraformaldehyde. The cornea was analyzed with fluorescent microscopic photographs or H&E staining.

Results: LPS induced TNF-α production was reduced by rBMP-7 with Hyaluronic acid or Cyclodextrin in J774A.1 cells. Cytotoxicity of Hyaluronic acid or Cyclodextrin did not show in HaCaT cells. The excipients supported formulation for eye drops due to viscosity and did not interfere with effects of recombinant BMP-7 in mouse alkali model.

Conclusions: Recombinant BMP-7 with Hyaluronic acid or Cyclodextrin has the ability to inhibit the inflammatory response and reduce fibrosis on a corneal alkali injury model in mice. Therefore, Hyaluronic acid or Cyclodextrin based rBMP-7 is available for eye drops and potential therapeutic reagents for eye scar therapy.

Keywords: 765 wound healing