Category: Formulation and Quality
Purpose: Age related macular degeneration (AMD) is one of the leading causes of blindness among elderly people in USA. Overexpression of vascular endothelial growth factor (VEGF) and pro-inflammatory mediators (TNF- α, IL-6, IL-1β, etc.) are responsible for the pathogenesis of choroidal neo-vascularization (CNV) in AMD. Celecoxib (CXB), a COX-2 inhibitor, provides numerous beneficial effects including anti-inflammatory, anti-VEGF, and anti-proliferative activities and may have the potential for the treatment of AMD. However, poor aqueous solubility and bioavailability limit application of CXB in ocular diseases. The purpose of this study is to develop a clear aqueous biotinylated nanomicellar drop formulation of CXB for back of the eye delivery utilizing self-assembly of hydrogenated castor oil (HCO-40) and biotinylated octoxynol-40 (OC-40) to treat AMD. Previous studies showed high expression of SMVT transporter at retinal and human corneal epithelial cells. Therefore, biotin was used a s a targeting moiety and conjugated with OC-40 with help of EDC/DMAP chemistry.
Methods: H-NMR and FT-IR were obtained to confirm the synthesis of biotinylated OC-40. RP-HPLC was used to determine entrapment efficiency and drug loading of biotinylated nanomicelles loaded CXB. Dynamic light scattering (DLS) was used to determine the size, PDI and zeta potential of our formulation. VEGF-A ELISA was used to determine the anti-VEGF activity of our formulation on retinal cells (D407). Proinflammatory cytokines such as TNF-α and IL-6 were determined with corresponding ELISA to assess the anti-inflammatory effect of the formulation on macrophage cells (RAW 264.7). Confocal laser microscopy was used to localize the nanomicelles.
Results: The weight ratio of 2.5: 0.01, HCO-40: biotinylated OC-40, was used which lead to the entrapment efficiency and drug loading of 99.3 and 3.67, respectively. DLS data revealed that the average nanomicellar size was 14.70 ± 0.07 (nm), a PDI of 0.146 ± 0.012, and zeta potential of nearly zero. VEGF-A ELISA suggested that CXB nanomicelles may significantly reduce VEGF release from D407 cell line in dose dependent manner where 30 µM CXB nanomicelles inhibited VEGF release from retinal cells by almost 50% of the control. Moreover, the basal TNF-α, and IL-6 secretion from macrophages cells (RAW 264.7) was very low around 30 and 5 pg/ml, respectively. Therefore, treatment of RAW 264.7 cells with 1 µg/ml LPS alone results in significant increase in cytokine production as compared to the control group. Surprisingly, the level of TNF-α and IL-6 were significantly decreased compared to LPS group after addition of CXB nanomicelles. Interestingly, blank nanomicelles which have no drug exhibited anti-TNF-α, and anti-IL-6 activities and this can be attributed to the presence of ricinoleic acid in HCO-40, the main fatty acid found in castor oil, which is known for its anti-inflammatory activity. CXB nanomicelles revealed a very significant reduction in inflammatory cytokines compared to blank nanomicelles which can be due to dual activities of CXB and ricinoleic acid that present in HCO-40 polymer. Confocal laser scanning indicated that biotinylated nanomicelles were efficiently internalized into conjunctival and retinal cells 2 folds higher than non-biotinylated nanomicelles. However, when cells were incubated with excess 1 mM biotin the uptake of biotinylated nanomicelle has dramatically reduced which can be due to competitive saturation of SMVT transporter proving that the formulations utilize SMVT transporter.
Conclusion: CXB loaded biotinylated nanomicelles were synthesize following solvent evaporation thin film rehydration technique. The ratio of 2.5: 0.01, HCO-40: biotinylated OC-40, was used with the entrapment efficiency and drug loading of 99.3 and 3.67, respectively. H-NMR and FT-R were used to analyze the purified product. The huge reduction of VEGF secretion from D407 cells after exposure to CXB nanomicelles proved the anti-VEGF activity of CXB nanomicelles. Furthermore, LPS induced TNF-α and IL-6 release from macrophages cells (RAW 264.7) was reduced upon addition of CXB nanomicelles proving the anti-inflammatory activity. Cellular internalization study was performed with confocal showed high cellular uptake of biotinylated nanomicelles loaded CXB compared to non-biotinylated one. Finally, biotinylated nanomicelles loaded CXB can be a promising delivery system for managing AMD, DME and DR.
Dhananjay Pal– University of Missouri, Kansas City, Kansas City, Missouri