title: Stability and in vitro toxicity of an infliximab eye drop formulation.
abstract: The purpose of this study was to develop a novel 10-mg/mL infliximab eye drop, to characterize its physical and biological stability under recommended storage conditions, and to assess the formulation's toxicity to ocular surface epithelium in vitro. Infliximab (10 mg/mL) was reconstituted using equal volumes of sterile water and 1% carboxymethylcellulose artificial tears. Aliquots were stored in either a 4 degrees C refrigerator or -20 degrees C freezer for up to 45 days. Physical stability was assessed through monitoring the solution's appearance, pH, ultraviolet-visible-near infrared absorbance and scattering, as well as protein gel electrophoresis. Biological stability was assayed through binding to tumor necrosis factor-alpha using an enzyme-linked immunosorbent assay. In vitro cytotoxicity to human corneal-limbal epithelial cells was examined following a 4-hour exposure to the study drug. Refrigerated and frozen infliximab eye drops remained clear and colorless for the duration of study. The formulation's pH (7.0) was comparable to that of the artificial tear vehicle alone. Low levels of ultraviolet-visible-near infrared light absorbance and scattering established the lack of protein precipitate after refrigeration or freezing. Protein gel electrophoresis performed under reducing conditions revealed the presence of two main protein bands of approximately 50 kDa and 25 kDa, representing immunoglobulin G heavy and light chains. The migration pattern of the proteins did not change under the different storage conditions and between day 10 and 45 after formulation. Infliximab binding to tumor necrosis factor-alpha remained stable for up to 45 days, with conservation of 101% and 102% of its initial binding activity when refrigerated or frozen, respectively. In vitro human corneal-limbal epithelial cultures showed no increase in cytotoxicity with infliximab treatment when compared to vehicle and culture media controls (P > 0.05). Infliximab can be formulated as an eye drop and remains stable when stored in accordance with current regulations regarding compounded eye drops. The demonstrated physical and biological stability as well as in vitro innocuity of this infliximab eye drop formulation may facilitate future clinical investigation targeting tumor necrosis factor-alpha as a modulator of various ocular surface diseases.
authors.affiliation: Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA. Marie-Claude_Robert@MEEI.harvard.edu
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Robert
authors.forename: Sandra
authors.initials: S
authors.lastname: Spurr-Michaud
authors.forename: Mathieu
authors.initials: M
authors.lastname: Frenette
authors.forename: David
authors.initials: D
authors.lastname: Young
authors.forename: Ilene K
authors.initials: IK
authors.lastname: Gipson
authors.forename: Claes H
authors.initials: CH
authors.lastname: Dohlman
mesh_terms: D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000737:chemistry* / Q000494:pharmacology / Q000633:toxicity*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002626:Chemistry, Pharmaceutical; D003315:Cornea / Q000187:drug effects; D004355:Drug Stability; D006801:Humans; D000069285:Infliximab; D009883:Ophthalmic Solutions / Q000008:administration & dosage / Q000737:chemistry* / Q000494:pharmacology / Q000633:toxicity*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors*
chemical_list: D000911:Antibodies, Monoclonal; D009883:Ophthalmic Solutions; D014409:Tumor Necrosis Factor-alpha; D000069285:Infliximab