Category: Preclinical Development
Purpose: Anti-VEGF monoclonal antibodies (mAb) are used in the intravitreal injection treatment of age-related macular degeneration (AMD) or diabetic retinopathy (DR). We developed a novel topical composition that enables transscleral permeation of bevacizumab, aflibercept and ranibizumab . The anti-aggregation eye drop formula (AAF) was first tested in vitro with human sclera . AAF retains mAbs in monomeric form and prevents loss of activity. Permeation studies, using human eye donor sclera as the model membrane, demonstrated mAbs compounded in AAF traversed eye tissue in appreciable quantities. The purpose of this study was to assess in vivo feasibility of delivering bevacizumab, an anti-VEGF mAb, to the back of the eye using AAF and administered as an eye drop.
Methods: Fifteen, 3-6 month old New Zealand F1 rabbits were designated Group 1 (bevacizumab in AAF, 5 mg/ml (0.5%), 6 rabbits); Group 2 (bevacizumab in PBS, 5 mg/ml (0.5%), 7 rabbits) and Vehicle control (AAF, 2 rabbits). Materials and methods, for the preparation of AAF and PBS, were previously described in Giannos et al . Hyaluronic acid (HA) 0.1% was added to AAF, PBS and AAF vehicle control to aid in mucoadhesion. AAF/HA and PBS/HA compositions were sterile filtered prior to addition of 5 mg/ml bevacizumab. AAF vehicle control was absent bevacizumab. Pharma grade components in AAF and PBS were used throughout and are approved for use in humans. Rabbits received an eye drop of approximately 100 µL in each eye, twice daily for 14.5 days, providing a total of 1 mg/day/eye. At the end of treatment, animals were anesthetized, serum collected and animals euthanized. Aqueous, vitreous, and retina, samples were collected into aliquots containing pre-weighed AAF composition as an assay diluent. Samples were stored at 4°C for 1-2 days prior to analysis. Quantitative ELISA was performed to analyze for, and determine the delivered concentration free bevacizumab.
Results: Table 1 shows the results from the in vivo rabbit study, using the full length antibody bevacizumab (Avastin®). The drug in AAF permeated and significantly accumulated in the aqueous, vitreous and retina; 10 times or more than when it was diluted in PBS. Additionally, our AAF/0.1% hyaluronic acid composition with 5 mg/ml bevacizumab, dosed 2 times a day (a clinically relevant dosing schedule) provided higher tissue concentrations in aqueous (1950%), vitreous (100%) and retina (62%) compared to prior work by Nomoto  using 5 times higher (25 mg/ml) concentrations dosed with a clinically unacceptable schedule (6 times/day). Studies of diseased human (AMD & DR), non-treated neovascular eyes, show a VEGF range of 0.1 to 0.7 ng/ml (100 to 700 pg/ml) in the vitreous [4,5]. Our eye drop delivery of bevacizumab into vitreous represents an achievable 1.3x in the stoichiometric ratio of drug to vascular endothelial growth factor (VEGF) concentration in vitreous for an untreated AMD or DR patient.
Conclusion: Bevacizumab, when diluted in AAF and administered topically, permeated and accumulated in rabbit aqueous, vitreous and retina at much higher concentrations, than when diluted in PBS under the same conditions. Our earlier in vitro human sclera permeation studies of mAbs in AAF showed ranibizumab, the fragment (Fab) of bevacizumab, had a 4 - 5x increase in flux compared to bevacizumab, which suggests further clinical relevance for an eye drop approach. A twice a day dosing of dilute anti-VEGF mAb drug would offer continuous and constant VEGF binding potential; providing a therapeutic level and much lower resident concentration of anti-VEGF mAb. Eye drops of anti-VEGF mAbs may provide better clinical efficacy compared to once a month bolus intravitreal injection.
Edward Kraft– Galveston, Texas
Praveena Gupta– Galveston, Texas
Jaafar El-Annan– Galveston, Texas
Mary Schmitz-Brown– Galveston, Texas
Valentina Reffatto– Galveston, Texas
Elizabeth Urias– Galveston, Texas
Kevin Merkley– Galveston, Texas