(T1230-09-60) Development of Long-Acting Griseofulvin Formulations for Local Treatment of Ocular Neovascularization

Purpose: Neovascular age-related macular degeneration (nAMD) is a progressive neurodegenerative ocular disease with limited drug options. Griseofulvin (GRF) is an FDA-approved antifungal drug that inhibits microtubule assembly. As an off-target effect, GRF metabolism produces a competitive analog of protoporphyrin IX (PPIX), a precursor to a cofactor of proangiogenic proteins, which interferes with angiogenesis [1]. Given the chronic nature of nAMD, long-acting formulations are essential to improving patient experience. The objective of the study is to develop sustained delivery systems of GRF to reduce its dosing frequency.

Methods: GRF-encapsulating poly(lactic-co-glycolide) (PLGA) microparticles (MPs) were prepared by the single emulsion method. Polymeric implants were prepared by hot-melt extrusion with five types of PLGA, differing in the lactide to glycolide (L:G) ratio, molecular weight, and end cap (Table 1). Target diameter of the implant was 500 µm, which can pass through a 23-gauge canula. The implants were produced at 120 °C with a screw speed of 20 rpm and a constant torque value of 0.2 Nm. MPs and implants were visualized with scanning electron microscopy. In vitro drug release kinetics were evaluated in phosphate buffered saline containing 0.2% Tween 80 (PBST). Implants were also analyzed by Powder X-ray Diffraction (PXRD), Fourier Transform Infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), and energy dispersive X-ray (EDX) spectroscopy.

Results: GRF MPs with a drug loading of 3-5 wt% inhibited the progression of neovascularization for 1.5 months in the mouse model of laser-induced choroidal neovascularization [2]. To extend the therapeutic activity to 3 or 6 months, we increased the GRF feed up to 25 wt%. When the target GRF loading exceeded 10 wt%, unencapsulated GRF was seen around the MPs in scanning electron microscopy images. This result suggests that the loading capacity of PLGA MPs is insufficient for extending the drug effect beyond 1.5 months. Alternatively, we produced GRF-loaded PLGA implants by hot melt extrusion. PLGA implants with a loading efficiency of 30 wt % had a diameter of 580 µm, sufficient to pass through a 23-gauge canula (Figure 1).  Scanning electron microscopy revealed differences in surface morphology - implants prepared with higher molecular weight PLGA (intrinsic viscosity 0.8-1.2 dL/g; L:G ratio 50:50) exhibited rough and porous morphology, whereas those with lower molecular weight PLGAs (intrinsic viscosity 0.32-0.44 dL/g; L:G ratio 50:50 and 75:25) had smoother surface. In the first 90 days, drug release rate showed some correlation with L:G ratio and end cap chemistry among the implants made of polymers from the same vendor. However, all implants, irrespective of the PLGA types, demonstrated near 0-order drug release kinetics in PBST that continued over 130 days (Figure 2), suggesting that drug release might be governed by additional mechanisms than polymer swelling and/or degradation. PXRD found GRF to be crystalline and PLGA amorphous. FT-IR and DSC indicated minimal interactions between the GRF and PLGA; i.e., two phase systems with crystalline GRF encapsulated in the amorphous PLGA matrix. Consistently, EDX analysis indicated the presence of GRF crystalline clusters in the PLGA matrix. These solid-state analyses suggest that drug release may be controlled by percolation, where drug release is dependent on the interconnecting pore channels formed upon previous drug release.

Conclusion: GGRF MPs with a drug loading of 3-5 wt% showed the sustained antiangiogenic effect over 1.5 months. However, further extension of drug effects was challenged by limited drug loading capacity. For higher drug loading, GRF PLGA implants were prepared by hot melt extrusion. The implant with 30 wt% drug loading showed < 10% initial burst release and continuous drug release for 130 days in PBST. The overall release kinetics were similar irrespective of polymer properties. Solid-state analyses suggest that drug release may be controlled by percolation following dissolution of encapsulated GRF crystals.

References: 1. Basavarajappa HD, Sulaiman RS, Qi X, Shetty T, Sheik Pran Babu S, Sishtla KL, Lee B, Quigley J, Alkhairy S, Briggs CM, Gupta K, Tang B, Shadmand M, Grant MB, Boulton ME, Seo SY, Corson TW. Ferrochelatase is a therapeutic target for ocular neovascularization. EMBO Mol Med. 2017 Jun;9(6):786-801.
2. Chobisa D, Muniyandi A, Sishtla K, Corson TW, Yeo Y. Long-Acting Microparticle Formulation of Griseofulvin for Ocular Neovascularization Therapy. Small. 2024 Mar;20(10):e2306479.

Acknowledgements: This work was supported by the National Institutes of Health (R01EY035159).

Figure 1. GRF PLGA implant placed in a non-valved 23-gauge canula

Table 1. PLGA polymers used to prepare the GRF PLGA implants

Figure 2. In vitro drug release of GRF PLGA implant in PBST. The ratio of griseofulvin to release medium was adjusted to achieve sink condition. n = 3, mean ± standard deviation.