(T1530-09-50) Development of an In Vitro Release Test to Characterize Onivyde Using Bio-Beads SM-2 Resin

Purpose: Onivyde, a pegylated irinotecan liposomal formulation, was approved by the U.S. Food and Drug Administration (FDA) in 2015 for patients with metastatic pancreatic adenocarcinoma who have previously undergone gemcitabine-based therapy. In 2023, Onivyde achieved annual sales of $156.3 million. A significant milestone was reached in June 2023 when the FDA accepted a supplemental New Drug Application, designating Onivyde as a first-line treatment for metastatic pancreatic ductal adenocarcinoma. With Onivyde's patents nearing expiration in 2025, generic pharmaceutical companies are increasingly interested in securing licenses for its generics, underscoring its substantial market value. However, the complex nature of liposomal formulations, due to their unique structure, poses significant challenges. Additionally, insufficient characterization of Onivyde's release behavior complicates these efforts. Therefore, this study aims to develop and validate an in vitro release test (IVRT) for Onivyde to characterize its in vitro release performance. The IVRT is crucial as a quality control procedure, enabling differentiation between various qualities of liposomal irinotecan products with differing release rates. The establishment of a robust IVRT is further essential for comparing in vitro release profiles of different formulations before initiating bioequivalence studies.

Methods: The IVRT for Onivyde was established using Bio-Beads SM-2 Resin (Biobeads), which are analytical-grade, nonpolar polystyrene adsorbents specifically designed for hydrophobic interaction chromatography. These macroporous polymeric beads have a high surface area that facilitates the adsorption of irinotecan when it is released from the liposomes, making them an effective tool for assessing in vitro release profiles. The release study was conducted using an incubated rotator set at predetermined rotational speed and temperature conditions. Onivyde was diluted with HEPES buffer to the desired concentration and mixed with a specified amount of Biobeads. Release samples were collected at specific time intervals, and the concentration of irinotecan in these samples was analyzed using a fluorescence spectrometer. Our experimental design involved systematically optimizing several parameters, including initial formulation concentration, the amount of Biobeads added, rotation speed and angle on the incubated rotator, temperature conditions, and agitation methods. This comprehensive exploration aimed to optimize and refine the IVRT for accurate characterization of Onivyde's in vitro release performance. Additionally, the particle size distribution was characterized using a Malvern Mastersizer.

Results: The amount of Biobeads added had a slight effect on the release profiles of Onivyde. Although a higher quantity of Biobeads resulted in an increased release rate, the differences in release profiles among the 100 mg, 200 mg, and 400 mg samples were not pronounced. A parallel study involving a free irinotecan solution provided insights into the adsorption kinetics of irinotecan by Biobeads upon its release from the liposomes. The adsorption efficiency exceeded 90% within 1 hour when using 200 mg of Biobeads, indicating rapid adsorption of the released drug. Under these conditions, over 90% of the drug was released from Onivyde within a 24-hour period, leading to the decision to utilize 200 mg of Biobeads in subsequent studies. The release profile of Onivyde was influenced by rotation speed; increasing the rotational speed resulted in a proportional increase in the release rate, highlighting its role in facilitating drug release. Furthermore, both particle size and polydispersity index (PDI) increased at higher rotation speeds, suggesting that the accelerated release rate at elevated speeds may be attributed to changes in particle morphology. Additionally, the investigation of release rates at different rotation angles revealed that a 90-degree rotation, which exerted the greatest force on the particles among all tested conditions, resulted in a faster release profile for Onivyde. This finding further confirmed that increased rotational force applied to the liposomes enhances the release rate. In Onivyde, three lipids constitute the lipid bilayer structure of the liposomes: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2k). The concentrations of these lipids were monitored throughout the release period to investigate whether drug release from Onivyde was influenced by the absorption of lipids within the lipid bilayer structure of the liposomes. The results indicated that both DSPC and DSPE-PEG2k remained present in the formulation during the release study. Cholesterol concentration has not yet been measured in the release samples.

Conclusion: In conclusion, this study establishes an in vitro release test (IVRT) for Onivyde using Bio-Beads SM-2 Resin (Biobeads), demonstrating its utility in characterizing the release behavior of liposomal formulations. Our results highlight that the release profile of Onivyde is influenced by Biobeads concentration, rotation speed, and angle. Importantly, the persistent presence of key lipids, such as DSPC and DSPE-PEG2k, during the release study suggests that drug release is not driven by lipid absorption. These findings not only contribute to the refinement of the IVRT for Onivyde, enhancing quality control, but also establish the assay as a reliable approach for evaluating generic irinotecan liposomal formulations.

Figure 1. Effect of Biobeads concentration 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. (A) Effect of rotation speed on the release profiles of Onivyde. (B) Z-average particle size of Onivyde throughout the release period. (C) Polydispersity index of Onivyde throughout the release period. (D) Effect of rotation angle on the release profiles of Onivyde. Data were presented as mean ± SD (n = 3).

Figure 3. Relative concentration of (A) DSPC and (B) DSPE-PEG2k in Onivyde throughout the release period. Data were presented as mean ± SD (n = 3).