This paper discusses the development and validation of a rapid method for the reversed phase HPLC-UV quantification of biodegradable poly(D,L-lactic-co-glycolic) acid (PLGA) microspheres co-loaded with two neuroprotective agents (dexamethasone and melatonin) (DX-MEL-MSs) to be intravitreally administered as a promising glaucoma treatment. The study was performed to validate two procedures that quantify the content of the two active substances entrapped into the polymer matrix during an encapsulation efficiency assay and the amount of drugs liberated over time during the in vitro release assay. The reversed-phase method allowed for the simultaneous determination of dexamethasone and melatonin, which were respectively detected at 240.5 and 222.7 nm. Chromatographic separation was performed using an Ascentis® C18 HPLC Column (25 cm × 4.6 mm, 5 µm) with an isocratic mobile phase composed of methanol-water (70:30, v/v) with 1.0 mL min−1 flow rate. The two procedures were validated analytically in terms of system suitability testing, specificity, linearity, precision, accuracy, sensitivity, and robustness. Both the validated procedures were applied to characterize DX-MEL-MSs and were found appropriate to quantify the drug quantities encapsulated and estimate their release profile over 10 days. The validation study designed in this work can be helpful for planning any other protocols that refer to the quantification of PLGA based drug delivery systems.
Although the pathogenesis of glaucoma is not completely understood yet, all patients diagnosed with this chronic disease are observed with neurodegeneration. Neuroprotective treatment will benefit from joint administration of active substances directed towards several therapeutic targets. Our study focuses on the hypothesis that a combination of anti‐inflammatory and antioxidant drugs, namely dexamethasone (DX), melatonin (MEL) and vitamin E (VE), loaded in biodegradable drug delivery devices (microspheres) can be advantageous as a long‐term therapeutic strategy that can achieve protection of visual function and avoid retinal cells death. We further evaluated whether drugs' stability and release properties changed or not after sterilization, a key process for ophthalmic formulations. Biodegradable poly (lactic‐co‐glycolic) acid (PLGA) microspheres (MS) were elaborated using the oil‐in‐water emulsion solvent evaporation technique. DX and MEL were added in a ratio 1/2:10 (DX/MEL:PLGA)(w/w) and a volume of 40 μl of VE was also included as an oily additive. After lyophilisation, MS were subjected to 60Cobalt radiation at 25 kGy (S‐MS) in dry ice. Before and after gamma‐irradiation, MS were characterized by means of internal and external morphology, particle size, encapsulation efficiency, thermal properties and in vitro release profiles. The chosen 38–20 μm MS exhibited pores inside and at a surface level respectively when observed at transmission and scanning electron microscopes, due to a slow evacuation of organic solvent from the internal to the aqueous phase for the oily nature of VE. MS showed high drug loadings of DX (56.61 ± 2.42 μg/mg MS) and MEL (33.97 ± 2.64 μg/mg MS), which remained unaltered for S‐MS, and followed a unimodal distribution of particle size (30.14 ± 1.03 μm for MS; 32.75 ± 1.46 μm for S‐MS). Differential scanning calorimeter thermograms revealed no endothermic transitions at DX and MEL melting points, thus indicating effective encapsulation. Regarding in vitro release profiles, both drugs in MS were released in a controlled fashion for more than 50 days (0.47 ± 0.03 μg/day for DX; 0.60 ± 0.04 μg/day for MEL). DX was released during a total of 120‐day follow‐up period (94.12 ± 0.68% of total DX encapsulated). Both compounds in S‐MS showed a higher burst release values (p < 0.001) in the first 7‐day period (93.21 ± 0.89% for MEL; 43.16 ± 0.75% for DX) compared to MS ones (81.35 ± 0.85% for MEL; 26.48 ± 1.24% for DX). Similarity factor (f2) statistically confirmed that changes in 35‐day release profiles for both the actives were due to sterilization (f2‐MEL = 40.47; f2‐DX = 39.10). The developed formulation proved potential usefulness as a possible long‐term neuroprotective therapy due to its four‐month in vitro co‐delivery. Additional studies will be performed for assessing in vitro toxicity and in vivo effectiveness of both the sterilized and no‐sterilized biodegradable drug delivery devices.
Purpose: Neuroprotectants can modulate cell survival and molecular pathways common to several inherited retinal degenerations (IRD). Among them, 7,8‐dihydroxyflavone (7,8‐DHF), a brain‐derived neurotrophic factor (BDNF) mimetic, proved to rescue vision in dyeucd6 zebrafish model of IRD by upregulating BDNF–TrkB signalling [1]. A novel 7,8‐DHF structural analogue, namely OPGG‐A2, demonstrated an improved ability in restoring visual capacity. Considering these results, this work aims at evaluating long‐release devices loaded with the two neuroprotectants as possible therapies for chronic retinal diseases. Methods: Biodegradable poly(lactic‐co‐glycolic)acid(PLGA) microspheres (MS) were prepared following the oil‐in‐water emulsion solvent evaporation technique. 7,8‐DHF and OPGG‐A2 were separately included in ratio 1:10 with PLGA(w/w). MS loaded with 7,8‐DHF (FMS) and with OPGG‐A2 (A2MS) were characterized in terms of production yield, internal and external morphology, particle size, encapsulation efficiency and in vitro release profile. Results: After sieving and freeze‐drying processes, 38–20 μm (38MS) and 20–10 μm (20MS) particle sizes were selected as suitable to fit conventional 30‐gauge needles for intravitreal administration. Both the granulometric fractions followed a unimodal distribution for both FMS and A2MS. Both FMS and A2MS demonstrated spherical shape without presence of crystal inside and on the 38MS and 20MS surfaces. FMS showed high encapsulation efficiencies (53.4 ± 3.4 μg/mg 38MS; 30.7 ± 12.2 μg/mg 20MS) as A2MS (58.8 ± 5.6 μg/mg 38MS; 61.3 ± 1.0 μg/mg 20MS). Both drugs were released in a sustained manner during 30 days after a high burst release under the experimental conditions specified (13.7 ± 1.4 μg/mg FMS‐38MS; 11.4 ± 3.8 μg/mg FMS‐20MS; 11.5 ± 2.2 μg/mg A2MS‐38MS; 23.1 ± 1.7 μg/mg A2MS‐20MS). Conclusions: The two formulations managed in vitro liberating both 7,8‐DHF and OPGG‐A2 for 1 month and could be interesting as prototypes for testing long‐term intravitreal neuroprotective therapies. References 1. Daly C, Shine L, Heffernan T, et al. A brain‐derived neurotrophic factor mimetic is sufficient to restore cone photoreceptor visual function in an inherited blindness model. Sci Rep 7, 11 320 (2017).
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