Pharmacokinetic sampling and bioanalysis
For PK sampling, serial blood and tear samples of EGF were collected at the scheduled times; pre-dose and 0.25h, 0.5h, 1h, 2h, 4h, and 12h at -1d and 1d, and pre-dose and 12h at 2d after dosing for dose groups of SAD study; pre-dose and 0.25h, 0.5h, 1h, 2h, 4h, and 12h at -1d, 1d and 14d, pre-dose at 2d, 8d, and pre-dose and 12h at 15d after dosing for dose groups of MAD study. Tear samples were collected from the marginal tear strip of the lower lid near the medial canthus by using disposable microcapillaries.
Serum and tear concentrations of EGF were analyzed by a validated enzyme linked immunoassay (ELISA) using a human EGF Quantikine ELISA kit (R&D systems, Minneapolis, MN, USA). Serum and tear samples were diluted appropriately in the Assay Diluent RD1-6. EGF calibration standards were prepared at the following concentrations: 3.91, 7.81, 15.6, 31.3, 62.5, 125, and 250 pg/mL. 200 μL of diluted sample or standard was added to the plate and incubated for 2 hours at room temperature.
After the incubation, the plates were washed three times with the wash buffer. 200 μL of human EGF conjugate was added to each well then incubated for 2 hours at room temperature. After the incubation, the plates were washed three times with the wash buffer. 200 μL of the substrate was added to each well and incubated for 20 minutes at room temperature. Then, 50 μL of reaction termination solution was added to each well to terminate the reaction. The subsequent absorbance was quantified by measurement at 450 nm using a VersaMax Microplate Reader (Molecular Devices, San Jose, CA, USA) and the results were analyzed using Softmax Pro 7.1 GxP (Molecular Devices, San Jose, CA, USA).