(M1430-10-57) Development of a Single-Vial Drug Release Assay Utilizing Bio-Beads SM-2 Resin to Evaluate Doxorubicin Liposomes

Purpose: Doxil® is a complex, liposomal doxorubicin hydrochloride formulation intended for parenteral administration to treat several cancer indications. The development of an in vitro drug release method for doxorubicin liposomes supports the physicochemical characterization necessary for bioequivalence and approval of generic formulations. Our lab has previously developed in vitro release methods for Doxil® and similar liposomal products such as Ambisome®, using a flow-through USP-4 dissolution apparatus. However, there is a lack of simple, cost-effective in vitro release assays available for the evaluation of liposomal doxorubicin formulations. We sought to develop a simple, robust, and reproducible IVR assay using Bio-Beads SM-2 adsorbent resin as sink material to support the regulatory assessment of complex liposomal generics using Doxil® as the model drug. Biobeads are nonpolar, macroporous polystyrene beads capable of absorbing hydrophobic and organic materials, and their use in pharmaceutical applications has not yet been extensively explored.

Methods: Three different Biobead weights (50, 100, and 200 mg) were studied to evaluate the effect of Biobead weight on doxorubicin release from liposomes in release media previously determined for doxorubicin liposomes by our lab for 24 hours at an assay temperature of 45⁰C. The release media (AMNS media) consisted of 100 mM NH₄HCO₃, 75 mM MES buffer, 5% w/v hydroxypropyl-β-cyclodextrin, 5% w/v sucrose, and 0.02% sodium azide at pH 6 [1]. A concentration study was performed in the same media to determine the optimal sample-in-media concentration for IVR assessment of doxorubicin liposomes. The contents of the release media were evaluated as individual components to study the individual effects of each component on doxorubicin release. 1X PBS pH 7.4 was then used to compare doxorubicin release from liposomes from physiologically relevant media using the same test conditions to evaluate doxorubicin release in AMNS media. Media optimization using 1X PBS pH 7.4 and different sources of cyclodextrin was performed to evaluate liposome integrity after drug release. Cumulative doxorubicin release was quantified in each study using a microplate-based spectrophotometer to quantify the fluorescent intensity of doxorubicin remaining in release media at specified time intervals (0, 1, 2, 4, 8, 12, 24 hours). Particle size distribution and polydispersity index were measured for each study condition at time points throughout each study using a Malvern ZetaSizer Nano. pH values were measured before and after each study.

Results: Bio-bead weight of 200 mg at a doxorubicin concentration of 10 µg/mL in release media yielded 91.7% doxorubicin release. In AMNS release media at pH 6 and 45°C, complete disintegration of the liposome was observed over 24 hours from time-dependent particle size distribution analysis. Evaluation of individual media components on doxorubicin release revealed that cyclodextrin was capable of preventing liposome disintegration while enabling doxorubicin release, however, at lower rates. Optimization of release media using 1X PBS pH 7.4 with hydroxypropyl-β-cyclodextrin indicated 10% w/v concentration was capable of inducing 93.7% doxorubicin release within 24 hours, respectively. The effect of different cyclodextrins at 5% w/v in 1X PBS pH 7.4 revealed that α-cyclodextrin, hydroxypropyl-β-cyclodextrin, γ-cyclodextrin, and dextrose were capable of inducing 87.1%, 80.1%, 98.64% and 100.03% doxorubicin release, respectively. However, liposome integrity was only retained for hydroxypropyl-β-cyclodextrin and α-cyclodextrin.

Conclusion: Development of this single-vial assay is useful as it can provide a simple means to assess in vitro drug release behavior of similar, liposomal products and present potential strategies to overcome challenges due to liposome structural integrity after drug release. Simplification of previously established release media reveals cyclodextrin as a principal excipient useful for the retention of liposome structural integrity after drug release which is critical for the evaluation and comparison of drug release kinetics for the development of complex, liposomal generics.

References: 1. Yuan, W.; Kuai, R.; Dai, Z.; Yuan, Y.; Zheng, N.; Jiang, W.; Noble, C.; Hayes, M.; Szoka, F. C.; Schwendeman, A. Development of a Flow-Through USP-4 Apparatus Drug Release Assay to Evaluate Doxorubicin Liposomes. AAPS J, 2017, 19 (1), 150–160. https://doi.org/10.1208/s12248-016-9958-2.
2. Tang, J.; Srinivasan, S.; Yuan, W.; Ming, R.; Liu, Y.; Dai, Z.; Noble, C. O.; Hayes, M. E.; Zheng, N.; Jiang, W.; et al. Development of a Flow-through USP 4 Apparatus Drug Release Assay for the Evaluation of Amphotericin B Liposome. European Journal of Pharmaceutics and Biopharmaceutics, 2019, 134, 107–116. https://doi.org/10.1016/j.ejpb.2018.11.010.

Acknowledgements: This research was supported by the Rackham Merit Fellowship awarded to Adaeze Eneli at the University of Michigan Rackham Graduate School. Anna Schwendeman declares financial interests for board membership, as a paid consultant, for research funding, and/or as equity holder in EVOQ Therapeutics and ASKO Pharma. The University of Michigan has a financial interest in EVOQ Therapeutics, Inc.