Biopharmaceutical Assessment of Dexamethasone Acetate-Based Hydrogels Combining Hydroxypropyl Cyclodextrins and Polysaccharides for Ocular Delivery

We previously developed two optimized formulations of dexamethasone acetate (DXMa) hydrogels by means of special cubic mixture designs for topical ocular administration. These gels were elaborated with hydroxypropyl-β-CD (HPβCD) and hydroxypropyl-γ-CD (HPγCD) and commercial hydrogels in order to enhance DXMa water solubility and finally DXMa’s ocular bioavailability and transcorneal penetration. The main objective of this study was to characterize them and to evaluate in vitro, ex vivo, and in vivo their safety, biopermanence, and transcorneal permeation. Gels A and B are Newtonian fluids and display a viscosity of 13.2 mPa.s and 18.6 mPa.s, respectively, which increases their ocular retention, according to the in vivo biopermanence study by PET/CT. These hydrogels could act as corneal absorption promoters as they allow a higher transcorneal permeation of DXMa through porcine excised cornea, compared to DEXAFREE® and MAXIDEX®. Cytotoxicity assays showed no cytotoxic effects on human primary corneal epithelial cells (HCE). Furthermore, Gel B is clearly safe for the eye, but the effect of Gel A on the human eye cannot be predicted. Both gels were also stable 12 months at 25 °C after sterilization by filtration. These results demonstrate that the developed formulations present a high potential for the topical ocular administration of dexamethasone acetate.

200,320 5678493 The standard calibration curves plotted the obtained mean peak area as a function of the concentration of DXMa are reported in Error! Reference source not found.2 for both Gels A and B.
The regression parameters of the lines are reported in Table S1. Slopes were significantly different from zero (p-value < 5%) and interceptions were not significantly different from zero (p-value > 5%). The determination coefficient (R²) value was found to be > 0.99. Hence, the method has linear response over the performed concentration range.

Accuracy (Bias %)
The accuracy studies were performed to verify the closeness of the agreement between the expected and the determined values. The DXMa concentration spiked in Gels A or B were determined using a linear regression. The accuracy was evaluated by calculating first the percentage recovery and then the percentage of relative standard deviation (RSD) of recovery. The recovery results obtained from the five standards of calibration levels were between 98.48 and 101.07% for DXMa in Gel A and between 98.40 and 101.01% for DXMa in Gel B. The values are within the limit of acceptance (95-105%). The RSD (%) of all five levels were 1.31% for DXMa in Gel A and 0.97% for DXMa in Gel B. The results were lower than the limit of acceptance (2%), indicating that the method is accurate.

Specificity
Specificity was examined by analyzing only the excipients of each gel (Gel A or B without DXMa). The absence of interference with DXMa was demonstrated (chromatogram not shown). In complement, to prove the specificity of the method, the degradation studies under relevant stress conditions were also performed and degradation products were observed after stress treatment ( Figures S3 and S4).  None of the observed peaks interfered with the DXMa peak in terms of retention time (resolution greater than 1.5). The used methods are therefore capable of identifying degradation products separately from DXMa. It should be noted that at this stage, we did quantify these degradation products.

Precision
Synthetic blend solutions representing 100 % of the target concentration of the method were used. The precision parameter was evaluated by performing both repeatability (intra-day variability) and intermediate precision (inter-day variability).
The repeatability characterizes the reproducibility of a given analytical procedure for the same sample preparation, as performed by the same analyst using the same instrument during a relatively short period time (intra-day). The repeatability was demonstrated by preparing six sample solutions (100%) measured by HPLC and calculating the relative percentage of standard deviation (RSD). For both formulations, the repeatability RSD values were 0.29% (Gel A) and 0.36% (Gel B). The RSD (%) values for intra-day are found to be < 2%, which were considered acceptable.
The intermediate precision characterizes the reproducibility of results obtained in the same laboratory during a prolonged period. It was established by preparing six assay sample solutions similar to repeatability (level 100%) injected into a HPLC system as per proposed method on 3 different days. The RSD (%) of assay results was calculated. The intermediate precision results are 0.44% for Gel A and 0.55% for Gel B. The RSD (%) values for inter-day precision were found to be lower than 2%, which indicates that method is also reproducible. The method was considered to be precise.

Limit of detection and limit of quantification
Detection and quantification limits are the lowest detectable and quantifiable concentration that a method can achieve (Table S2). As per ICH guideline, the LOD and LOQ were determined based on the standard deviation of the response () and the slope (s) in accordance with the equations: LOD = 3.3 × /s and LOQ = 10 x /S. In conclusion, the chromatographic method described was validated for quantitative assay determination of DXMa in Gels A and B as per ICH Q1A (R2) guideline.
The developed method is specific, accurate, precise, and reproducible. All the degradation products formed during stress conditions were well separated from the DXMa peak demonstrating that the developed method was specific. The method, according to international guidelines, can be used to determine DXMa content over time since no interference with degradation products was observed.  Gel B