(T1030-06-32) Characterization and Quantification Analysis of Onivyde Irinotecan Liposome Injection

Purpose: Onivyde is an intravenous irinotecan liposomal injection approved by the FDA for post-gemcitabine metastatic adenocarcinoma of the pancreas. In the Onivyde formulation, irinotecan is encapsulated in the inner compartment of the liposome by a trapping agent, sucrose octasulfate, and stabilized by a pegylated lipid membrane, resulting in a prolonged circulation. Given its complex liposomal formulation, there is limited information on the critical quality attributes (CQAs) of Onivyde and appropriate methods to measure these CQAs. In the present work, we developed a series of analytical methods to characterize particle size, morphology, drug and lipid contents of Onivyde and its in vitro and in vivo release behavior.

Methods: Onivyde was purchased from the University of Michigan Health System Research Pharmacy. The particle size was measured by dynamic light scattering (DLS) using a Malvern ZetaSizer Nano ZS and the morphology of Onivyde was visualized by a cryogenic transmission electron microscope (cryo-TEM). The quantity of irinotecan was determined using ultra performance liquid chromatography-fluorescence detector (UPLC-FLR). To quantify the encapsulated irinotecan percentage in Onivyde, a TOYOPEARL HW-55F column was used to separate free drug from the liposome encapsulated drug. Lipid components including distearoylphosphatidylcholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2k) and cholesterol were separated using a Waters Xbridge C18 column and quantified by UPLC-evaporative light scattering detector(ELSD). In vitro drug release was performed with a SpectraPor Float-A-Lyzer dialysis device at 55℃ in 10mM phosphate buffer saline (PBS) with different concentrations of ammonium bicarbonate (0 mM, 0.1 mM, 0.5 mM NH₄HCO₃). Furthermore, a pharmacokinetic study was conducted in BALB/c mice, with a 15 mg/kg intravenous injection of either Onivyde or free irinotecan. The concentration of irinotecan in vivo was quantified by UPLC-MS. Particularly, free irinotecan in the plasma of Onivyde-dosed mice was separated by Waters Corp Oasis HLB Cartridge to analyze the in vivo drug release behavior. Graphs were generated using GraphPad Prism 8.

Results: Onivyde liposome exhibited an intensity-weighted mean particle size of 110 nm (Figure 1. A and F). In cryo-TEM images, Onivyde presented as a prolate spheroid or a spheroid, and irinotecan existed as an electron-dense precipitate in the core of liposome (Figure 1. D and E). Irinotecan and lipid contents matched well with the value stated in the package insert (Figure 1. F). In vitro, the cumulative drug release of Onivyde was less than 20% in pure PBS after 24 h, while it increased to 50% with the addition of 0.1 mM NH₄HCO₃ and reached 100% with 0.5 mM NH₄HCO₃ (Figure 2). In vivo, the half-life of irinotecan was significantly improved from 0.4 h to 3.0 h by the liposome formulation and the area under the curve of irinotecan from Onivyde-dosed mice was more than 200-fold to that from mice dosed with the free drug.

Conclusion: We established a series of analytical methods to characterize Onivyde in terms of particle size distribution, morphology, drug and lipid contents and its release behavior in vitro and in vivo. The characteristics and quantification analysis of Onivyde paved the way for further evaluation of lot-to-lot variability of approved product and development of generic versions of liposomal irinotecan.

Figure 1. Characterization and quantification of Onivyde. A. Particle size distribution of Onivyde (intensity-weighted results). B. Chromatograms of two forms of irinotecan. C. Chromatograms of lipids in Onivyde. D, E. Cryo-TEM images of undiluted Onivyde, scale bar represents 500 nm. F. Important parameters of Onivyde. All values are presented as mean ± SD (n=4).



Figure 3. Pharmacokinetic study results. A. Concentration of irinotecan (determined as total drug) in plasma after administration of free drug and Onivyde in mice. B. Concentration of free irinotecan in plasma of Onivyde-dosed mice C. Main pharmacokinetic parameters (determined as total drug) calculated by Phoenix WinNonlin software. All values are presented as mean ± SD (n=4).



Figure 2. Cumulative release of irinotecan from free drug or Onivyde in PBS with different concentrations of NH4HCO3.