Category: Formulation and Quality
Purpose: The primary purpose of the study was the development of a topical eye drop treatment of diabetic retinopathy which can serve as a possible alternative to the currently established intravitreal anti-VEGF therapy. Due to the chronic nature of Diabetic Retinopathy (DR) and the high integrity of the ocular barriers, monthly intravitreal injections of anti-VEGF antibodies are prescribed. Such intravitreal injections are not only painful for the patients but also can cause retinal hemorrhage. Thus topical eye drops of potent immunosuppressive agent, tacrolimus was developed using nanomicellar technology for the treatment of back-of-the eye disorder like DR. Tacrolimus nanomicellar formulation and optimization was carried out. This was followed by in-vitro evaluation of the safety and efficacy of the formulation in various ocular cell lines
Methods: Tacrolimus nanomicellar (TAC NMF) was prepared by solvent evaporation and film rehydration method using a mixture of amphiphilic polymers like Hydrogenated castor oil-40 (HCO-40) and Octyxonyl-40 (OC-40). A full factorial design of experiment was employed using student version of JMP 10.0® software. Characteristics of the self-assembling tacrolimus nanomicelles such as hydrodynamic size, PDI and zeta potential were determined. Tacrolimus (TAC) loading and entrapment efficiencies were determined using reverse phase HFLC method. Concentration-dependent in-vitro cytotoxicity was determined in conjunctival (CCL 20.2) and retinal pigment (D407) epithelial cells and human retinal endothelium (RF/6A) cell lines using MTT and LDH assay. Time-dependent in-vitro cellular uptake of TAC NMF was determined in CCL 20.2, D407 and RF/6A cell lines using confocal laser scanning microscopy (CLSM) and flow cytometry (FACS). In-vitro transwell diffusion assay was carried out by seeding CCL 20.2 and D407 cells in the upper and the lower chamber of the transwell plate respectively. Time-dependent uptake of TAC NMF added in the upper chamber was analyzed using FACS by collecting the bottom chamber cells. This represented an in-vitro model of the human eye. For the evaluation the biocompatibility of the formulation, macrophage cells (RAW 264.7) were utilized. ELIZA was used to analyze the production of inflammatory cytokines like IL-6, IL-1β and TNF-α after treating RAW 264.7 cells with TAC NMF. The in-vitro efficacy of the formulation was established by treating all the three ocular cell lines with sodium iodate (SI) for 6 hours followed by treatment with TAC NMF for 12 and 24 hours. The liberated IL-6, IL-1β and TNF-α and VEGF-A in the supernatant was evaluated by ELIZA.
Results: An aqueous, clear TAC NMF was prepared and optimized. TAC NMF had a mean diameter of 15.41 nm, a polydispersity index of 0.25 and a zeta potential of 0.5 mV. Entrapment and loading efficiencies of the formulation were 97.13% and 0.72% respectively. Drug encapsulation was confirmed by H1NMR. In-vitro cytotoxicity studies such as MTT assay and LDH assay revealed dose dependent cytotoxicity of TAC NMF on ocular cell lines. While the cell viability was almost 100% for the blank formulation indicating the high safety profile of the polymers HCO-40 and OC-40 used in this nanomicellar formulation. Cellular uptake studies using FACS and CLSM indicated an increase in uptake of TAC NMF in a time-dependent manner as compared to the drug TAC. Nanomicelles owing to their small size and neutral zeta potential get absorbed into the ocular cells by passive diffusion and can enhance absorption of highly hydrophobic drug like tacrolimus. In-vitro dual chamber transport studies depicted high transport of the TAC NMF in a time dependent manner. This indicated that nanomicelles can also transverse through the paracellular route through cellular layers. This can be helpful for the passive transport of nanomicelles from the anterior segment of the eye to the posterior segment through the trans-scleral pathway. In-vitro biocompatibility assay using macrophage RAW 264.7 resulted in no statically significant production of inflammatory cytokines like IL-6, IL-1β and TNF-α after treatment with TAC NMF for 24 hours. This indicated the biocompatibility of TAC NMF in-vitro. TAC NMF and TAC 12 and 24 hour treatment could lower the production of IL-6, IL-1β, TNF-α and VEGF-A in all the three ocular cell lines pretreated with SI. This implied that TAC NMF could lower the inflammatory response commonly seen in ocular tissues with DR.
Conclusion: A clear, aqueous, stable tacrolimus nanomicellar formulation was formulated and evaluated for its effectiveness. The potential of nanomicelles to reach back-of-the-eye coupled with a strong immunosuppressive effect of tacrolimus can prove to be a beneficial strategy to evade the painful intravitreal injections for DR.
Dhananjay Pal– University of Missouri, Kansas City, Kansas City, Missouri