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).