The availability of drug by liposomal drug delivery : Individual kinetics and tissue distribution of encapsulated and released drug in mice after administration of PEGylated liposomal prednisolone phosphate.

Lately, the usefulness of liposomal drug delivery systems has been debated. To better understand the underlying pharmacokinetics of the targeted drug delivery by liposomes, individual encapsulated and non-encapsulated drug concentrations in blood, tumor, liver, spleen and kidneys were quantified after i.v. administration of liposomal prednisolone phosphate in mice. Kinetic analysis shows that the tumor influx of encapsulated drug is not dominant compared to the uptake by the other tissues. Further, from a quantitative point of view, the availability of non-encapsulated drug in the tumor tissue after liposomal delivery is not pronounced as compared to the other tissues studied. However, drug release in the tumor seems more extended than in the other tissues and the non-encapsulated drug concentration decreases more slowly in the tumor than in the liver and spleen. The spleen shows a high affinity for the uptake of encapsulated drug as well as the release of drug from the liposomes. Subsequently, released drug in the spleen, and possibly also in other tissues, is probably quickly redistributed towards the blood and other tissues. This also impairs the drug delivery effect of the liposomes. In contrast to the released drug in the central circulation, liver and spleen, the released drug concentration in the tumor remains at a fairly constant level likely due to the extended release kinetics from the liposomes. These extended release characteristics in the tumor most probably contribute to the beneficial effect. Nevertheless, it should be noted that larger released drug concentrations are formed in healthy tissues.

Quantitative LC-MS determination of liposomal encapsulated prednisolone phosphate and non-encapsulated prednisolone concentrations in murine whole blood and liver tissue.

The underlying pharmacokinetic profile of liposomal drug delivery systems is not yet fully known. This is primarily due to a lack of suitable quantitative bioanalytical methodology to simultaneously determine separate liposomal-encapsulated and non-encapsulated drug tissue concentrations in complex biological samples. Here, an LC-MS method was developed which enables the simultaneous quantification of separate liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone concentrations in whole blood and liver tissue. Liquid chromatography, negative electrospray ionization and Orbitrap-MS analysis allowed highly accurate and sensitive detection of prednisolone phosphate (PP) and prednisolone (P) in complex matrix. Using dexamethasone phosphate and dexamethasone as internal standards, the quantitative LC-MS method was optimized and validated for high selectivity, sensitivity and quantitative accuracy of PP and P from liposomes. The lower limits of quantitation were 0.99µmol/L blood and 0.53nmol/g liver for PP, and 229nmol/L blood and 0.514nmol/g liver for P. Quantitative accuracies of 84-118% were observed. The intra-run precision was ≤11%. Application of this new LC-MS method will yield the first liposomal pharmacokinetic profile showing accurate encapsulated and non-encapsulated drug tissue concentrations separately. This is also the first quantitative LC-MS method for the simultaneous quantification of the prodrug PP and its parent drug P in whole blood and liver tissue samples.

Plasma, blood and liver tissue sample preparation methods for the separate quantification of liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone.

Besides the development of sample preparation methods for the determination of separate liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone concentrations in murine plasma and blood, this article also presents the first description of an accurate sample preparation method for the determination of such separate concentrations in the murine liver. The quantitative differentiation is based on the immediate hydrolysis of prednisolone phosphate (PP) into prednisolone (P) after its release from the liposomes in vivo: PP represents the encapsulated drug, while P represents the non-encapsulated drug. The use of 10 ml methanol/g tissue during homogenization of liver tissue ensures complete liposome rupture, prevention of the dephosphorylation of PP released during homogenization, sufficient clean supernatants, excellent extraction of P and sufficient extraction of PP and excellent accuracies and precision complying with the internal guidelines for pre-clinical studies (80-120% and maximal 20%, respectively). Similarly, the matching sample preparation methods for plasma and blood involve protein precipitation with four equivalents of methanol also ensuring accuracies and precision complying with the internal guidelines for pre-clinical studies. Application of these sample preparation methods is going to generate the first pharmacokinetic (PK) profile of a liposomal preparation, in which the encapsulated and non-encapsulated drug concentrations in a tissue are measured separately. Such separated concentration profiles can gain important insights into the PKs of liposomal PP and probably also with regard to liposomal formulations in general, like the quantification of the in vivo drug release from the liposomes.

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

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.

The development of a method to quantify encapsulated and free prednisolone phosphate in liposomal formulations.

This paper presents the development of a new method for the simple and reliable quantification of the free drug amount in liposomal preparations of prednisolone phosphate (PP). In this method the free drug is distinguished from the encapsulated drug by means of hydrolysis of the free PP into prednisolone (P) by alkaline phosphatase (AP). During method development reaction progress curves were recorded to determine the required AP concentration and the corresponding incubation time to achieve hydrolysis of all free PP. Reaction progress curves also showed that small changes in the amount of weighted AP and the incubation periods used do not cause a change in outcome. Further, several organic solvents were tested as precipitation solvent and the use of tetrahydrofuran (THF) yielded clean chromatograms, rapid AP deactivation and complete liposome rupture avoiding under- and overestimations of the encapsulated and free drug concentrations. Method accuracy was evaluated during a cross-validation involving dialysis. Intra- and interday precision were evaluated by determining the standard deviation (SD) and relative standard deviation (RSD) after applying the new method on one day (n=4) and on different days (n=3). The accuracy of the developed method is comparable to the accuracy determined by dialysis, while clearly the method using AP is more precise. In conclusion, comprehensive method development yielded an accurate and precise method, which can replace traditional methods like dialysis and solid phase extraction (SPE). With little effort the method can be upgraded and become part of the liposome certification prior to human use. The overall principle behind the method offers possibilities for many drug carrier systems.