(T0930-03-18) Development and Optimization of an In Vitro Release Assay for Evaluation of Liposomal Irinotecan Formulation

Purpose: Onivyde®, an FDA-approved liposomal irinotecan, has achieved significant market success with annual sales reaching $139.5 million. However, with its US patent exclusivity set to expire in 2025, the development of generic versions has become an attractive prospect. Nevertheless, meeting the stringent comparability criteria set by the FDA for generic irinotecan liposomal products poses considerable challenges. A key challenge arises from the insufficient characterization of Onivyde®'s release behavior in existing literature. Therefore, the aim of this study is to develop an in vitro release (IVR) assay for Onivyde® by investigating its release behavior under various conditions, including modifications in media composition, pH, temperature, irinotecan concentration, and dialysis bag type. By analyzing these release profiles, our study seeks to gain a deeper understanding of the factors influencing the IVR of Onivyde®. Moreover, we optimize the IVR assay to effectively differentiate between varying qualities of irinotecan liposomal formulations. The developed IVR assay holds potential for facilitating quality control of liposomal irinotecan and expediting the development and approval of generic products.

Methods: The release behavior of irinotecan from Onivyde® was investigated using a dialysis method with gentle shaking. To establish an IVR assay, various factors affecting its performance were examined, including media composition, temperature, irinotecan concentration, dialysis bag properties, and pH of the media. The NH₄⁺ concentration in the release media varied from 0 to 50 mM. The study encompassed three temperature conditions: room temperature (RT), 37°C, and 60°C, the latter exceeding the transitional temperature of the major lipid (DSPC) in Onivyde®. The final concentration of irinotecan in the release media ranged from 0.45 to 45.70 μg/ml. Different dialysis bags with varying molecular cut-off (MWCO) composed of regenerate cellulose (RC) and cellulose ester (CE) were employed. Additionally, specific buffers were utilized to achieve the desired capacity in the release media. A control group consisting of free irinotecan solution was included for comparison. The released irinotecan samples were quantified using a UPLC, and the cumulative release percentage was calculated by comparing the levels of irinotecan in the release samples with those in the control group. Subsequently, this optimized IVR assay was utilized to evaluate several different quality samples of Onivyde® and investigate its ability to discriminate between them. Specifically, the Onivyde® samples underwent treatments involving freeze-thaw cycles, heat, and mechanical stress to generate varying qualities of irinotecan liposomal formulations. Furthermore, the size distribution of the particles was characterized using a Malvern Mastersizer®.

Results: The release profile of Onivyde® was significantly influenced by the concentration of ammonium bicarbonate. Increasing the concentration of ammonium bicarbonate resulted in a proportional increase in the release rate, indicating its role in facilitating drug release. However, the presence of ammonium bicarbonate did not have any effect on the release profiles of the free irinotecan solution. The release rate of Onivyde® was accelerated at higher temperatures, with a notable enhancement compared to RT. Importantly, the size and polydispersity index of the liposomes remained stable under different temperature incubations. We observed that higher concentrations of irinotecan led to a slower release profile. Additionally, the release profiles were influenced by the MWCO of the dialysis bag, with differences observed using the CE membrane dialysis bags but not the RC membrane dialysis bags. Furthermore, when the pH of the release media was adjusted using different buffers, samples from higher pH release media exhibited a faster release profile, indicating the influence of pH on the release behavior of Onivyde®. The optimized IVR assay, which utilized a 50 kDa RC membrane in a medium containing 5 mM ammonium bicarbonate in HEPES buffer (pH 7.4) at 37°C, was successfully developed. This optimized IVR assay was then applied to stressed Onivyde® samples. While the particle distribution of the stressed Onivyde® samples remained relatively unchanged compared to intact Onivyde®, the release profiles of the stressed samples exhibited a faster release. This demonstrates the potential use of the optimized IVR assay as a reliable tool for assessing the release behavior and distinguishing between different qualities of irinotecan liposomal formulations.

Conclusion: In conclusion, the release of Onivyde® is influenced by key factors including ammonium bicarbonate concentration, temperature, irinotecan concentration, dialysis bag properties, and pH of the release media. The developed IVR assay is a reliable tool for assessing Onivyde® release behavior and distinguishing different qualities of irinotecan liposomal formulations. These findings contribute to optimizing IVR assays for liposomal irinotecan formulations, enabling better quality control procedures. The assay serves as a reliable tool for evaluating generic irinotecan liposomal formulations, aiding in their development and ensuring in vitro comparability.

Figure 1. Effect of ammonium bicarbonate concentrations on the release profiles of (A) Onivyde® and (B) free irinotecan solution at 37°C. The experimental data were presented as mean ± SD (n = 3).

Figure 2. Impact of (A) dialysis bag properties, (B) temperature, (C) irinotecan concentration, and (D) pH of release media on Onivyde® release profiles. The experimental data were presented as mean ± SD (n = 3).

Figure 3. In vitro release profiles of Onivyde® stressed by different methods. The experimental data are presented as mean ± SD (n = 3).