In vitro confirmation of the quantitative differentiation of liposomal encapsulated and non-encapsulated prednisolone (phosphate) tissue concentrations by murine phosphatases

Abstract

The quantitative differentiation of liposomal encapsulated and non-encapsulated drug tissue concentrations is desirable, since the efficacy and toxicity are only related to the level of non-encapsulated drug. However, such separate concentration profiles in tissues have still not been reported due to lacking analytical methodology. The encapsulation of prodrugs like prednisolone phosphate (PP) in liposomes offers new, analytical opportunities. Instantaneous dephosphorylation of PP into prednisolone (P) by phosphatases after its release from the liposome in vivo makes it possible to differentiate between the encapsulated and the non-encapsulated drug for such preparations of liposomal PP: PP represents the encapsulated drug, while P represents the non-encapsulated drug. In the here described study, the instantaneous dephosphorylation of PP by murine liver and kidney phosphatases has been verified by incubation of PP in liver and kidney homogenates followed by estimation of the dephosphorylation rate constants k and the dephosphorylation time of the expected maximal in vivo non-encapsulated drug concentrations. In vitro PP has been rapidly converted into P in the presence of homogenate from the excretory organs. The calculated values for k have shown that the liver contains more active sites per gram of tissue than the kidneys. However, the dephosphorylation of PP by these active sites is slower compared with the kidneys. Compared with other pharmacokinetic processes of P, the estimated dephosphorylation times of the expected maximal in vivo non-encapsulated drug concentrations in the liver and the kidneys are considered to be instantaneous. This enables the separate determination of the encapsulated and non-encapsulated drug concentrations in the excretory organs after administration of liposomal PP in mice generating the first pharmacokinetic profile of a liposomal preparation, in which the in vivo encapsulated and free drug tissues concentrations are measured separately. This can also gain important insights into the pharmacokinetics of liposomal formulations in general.

• Bioanalysis
• hydrolysis
• liposomes
• pharmacokinetics
• prodrug

Acknowledgements

Jon Curtis and Niels Kruize from KBioscience are acknowledged for their assistance during the application of the Covaris. The authors thank Hester Kramer from MSD Oss BV for reviewing this article. The authors acknowledge MediTrans for the financial support.

Notes

¹Here, free drug is defined as the non-encapsulated drug including non-protein-bound drug as well as protein-bound drug.

²In vivo data presenting the blood and tissue concentration--time profiles of total, encapsulated and free drug after intravenous administration of liposomal prednisolone phosphate in mice is being prepared for publication elsewhere.