A novel quantification method for unencapsulated doxorubicin in tissues: unlocking PK/PD and TK/TD insights for...

Pegylated liposomal doxorubicin (PLD) is the first FDA-approved liposomal product in the oncology field and has been extensively investigated over the years. Although PLD has been extensively studied, measurement of unencapsulated doxorubicin in tissues remains technically challenging, limiting detailed interpretation of tissue pharmacokinetics/pharmacodynamics (PK/PD) and toxicokinetics/toxicodynamics (TK/TD) relationships. Here, we present a novel approach to quantify unencapsulated doxorubicin in tumors and other tissues. Our method employs the gentleMACS Dissociator for tissue homogenization to prevent liposome disruption. While homogenization with conventional zirconia beads increased the unencapsulated doxorubicin fraction by 4.2- to 13.3-fold, homogenization with the gentleMACS Dissociator resulted in no increase in unencapsulated doxorubicin, demonstrating superior preservation of PLD integrity compared with conventional techniques. Moreover, homogenization of freeze-thawed tissue samples did not induce doxorubicin release, enabling sample cryopreservation prior to analysis. Using this approach, we observed a 3.7-fold higher fraction of unencapsulated doxorubicin relative to total (encapsulated + unencapsulated) concentration in tumors than in liver. This difference may reflect enhanced release and/or prolonged retention of released doxorubicin in tumors, as well as more rapid elimination of released doxorubicin in the liver. Our method is expected to provide critical insights into doxorubicin release from liposomes and the retention of released doxorubicin thereby facilitating the PK/PD and TK/TD analysis of liposomal products. By addressing a fundamental need in liposomal analysis, our method holds significant promise for accelerating drug development and improving cancer therapy outcomes.