Corneal toxicity of cell-penetrating peptides that inhibit Herpes simplex virus entry

Cell-penetrating peptides (CPPs) inhibit Herpes simplex virus entry at low micromolar concentrations and may be useful either as prophylactic or therapeutic agents for herpetic keratitis. The aim of this study was to assess the in vitro and in vivo toxicity of three CPPs-EB, TAT-C, and HOM (penetratin)-for the cornea. Incubation of primary (HK320) or immortalized (THK320) human keratocytes with the EB peptide (up to 100 μM), bHOMd (up to 200 μM), or TAT-C (up to 400 μM) resulted in no evidence of toxicity using a formazan dye-reduction assay. Similar results were obtained with a human trabecular meshwork cell line (TM-1), primary human foreskin fibroblasts (DP-9), Vero, and HeLa cells with EB and TAT-C. The bHOMd peptide showed some toxicity in Vero and HeLa cells, with CC₅₀ values of 70 and 93 μM, respectively. The EB peptide did not inhibit macromolecular synthesis in Vero cells at concentrations below 150 μM, although cell proliferation was blocked at concentrations of EB above 50 μM. In vivo toxicity was assessed by applying peptides in Dulbecco's modified Eagle's medium to the cornea 4 times daily for 7 d. At concentrations 1000 times the IC₅₀ values, the EB and bHOM peptides showed no toxicity, whereas TAT-C caused some mild eyelid swelling. Some slight epithelial cell sloughing was seen with the bKLA peptide in vivo. These results suggest that these CPPs-and EB in particular-have a favorable toxicity profile, and that further development is warranted.