Administration of Menadione, Vitamin K3, Ameliorates Off-Target Effects on Corneal Epithelial Wound Healing Due to Receptor Tyrosine Kinase Inhibition.
OBJECTIVE
The antiangiogenic receptor tyrosine kinase inhibitor (RTKi), 3-[(4-bromo-2,6-difluorophenyl)methoxy]-5-[[[[4-(1-pyrrolidinyl) butyl] amino] carbonyl]amino]-4-isothiazolecarboxamide hydrochloride, targets VEGFR2 (half maximal inhibitory concentration [IC50] = 11 nM); however, off-target inhibition of epidermal growth factor receptor (EGFR) occurs at higher concentrations. (IC50 = 5.8 μM). This study was designed to determine the effect of topical RTKi treatment on EGF-mediated corneal epithelial wound healing and to develop new strategies to minimize off-target EGFR inhibition.


METHODS
In vitro corneal epithelial wound healing was measured in response to EGF using a transformed human cell line (hTCEpi cells). In vivo corneal wound healing was assessed using a murine model. In these complementary assays, wound healing was measured in the presence of varying RTKi concentrations. Immunoblot analysis was used to examine EGFR and VEGFR2 phosphorylation and the kinetics of EGFR degradation. An Alamar Blue assay measured VEGFR2-mediated cell biology.


RESULTS
Receptor tyrosine kinase inhibitor exposure caused dose-dependent inhibition of EGFR-mediated corneal epithelial wound healing in vitro and in vivo. Nanomolar concentrations of menadione, a vitamin K3 analog, when coadministered with the RTKi, slowed EGFR degradation and ameliorated the inhibitory effects on epithelial wound healing both in vitro and in vivo. Menadione did not alter the RTKi's IC50 against VEGFR2 phosphorylation or its inhibition of VEGF-induced retinal endothelial cell proliferation.


CONCLUSIONS
An antiangiogenic RTKi exhibited off-target effects on the corneal epithelium that can be minimized by menadione without deleteriously affecting its on-target VEGFR2 blockade. These data indicate that menadione has potential as a topical supplement for individuals suffering from perturbations in corneal epithelial homeostasis, especially as an untoward side effect of kinase inhibitors.
Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky, United States.
Jamie S
JS
Rush
PanOptica, Inc., Bernardsville, New Jersey, United States.
David P
DP
Bingaman
PanOptica, Inc., Bernardsville, New Jersey, United States.
Paul G
PG
Chaney
PanOptica, Inc., Bernardsville, New Jersey, United States 3Department of Ophthalmology and Visual Sciences, Rutgers New Jersey Medical School Newark, New Jersey, United States.
Martin B
MB
Wax
Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky, United States 4Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, Kentucky, United States.
Brian P
BP
Ceresa
D000818:Animals; D002460:Cell Line; D004195:Disease Models, Animal; D004815:Epidermal Growth Factor / Q000378:metabolism; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D024483:Vitamin K 3 / Q000494:pharmacology*; D014945:Wound Healing / Q000502:physiology
D047428:Protein Kinase Inhibitors; D004815:Epidermal Growth Factor; D024483:Vitamin K 3; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2