Distribution of chemotherapeutic agents such as Doxorubicin and Paclitaxel into untargeted organs causes toxicity. Non liposomal Doxorubicin formulation causes myocardium damage that is cumulative and irreversible. In clinical practice, the cumulative dose of Doxorubicin must be limited to reduce cardiotoxicity. The development of chemotherapeutic agents to effectively treat solid tumors depends on the ability of these agents to achieve cytotoxic drug exposure within the tumor(s). Encapsulating common anticancer agents into nanoparticle delivery systems, particularly liposomes, provides a promising approach to enhance delivery at target site.
Liposomes are phospholipid bilayer vesicles, which can be used as delivery vehicles for chemotherapeutics. Liposomal Doxorubicin formulations have shown significant therapeutic advantages of lower toxicity and equivalent or higher anti-tumor effects compared to free Doxorubicin formulations. Because only free Doxorubicin has toxicological and pharmacological effects, understanding its pharmacokinetics can potentially lead to relatively precise prediction of the toxicity and efficacy of liposomal doxorubicin in vivo.
To understand the efficacy and toxicity of liposomal drugs, it is essential to establish a methodology for separation and determination of liposomal and non-liposomal (free) doxorubicin present in circulating blood through its pharmacokinetic study.
Here, we describe a methodology for extracting and quantifying free and liposomal Doxorubicin in the plasma of rats treated with liposomal Doxorubicin.
Two approaches are in use to quantify non-liposomal fractions of the formulations
Using Solid Phase Extraction
To separate liposomal and non-liposomal (free) doxorubicin the following procedure can be used. Separation of liposomal and non-liposomal (free) doxorubicin can be achieved by passing the plasma/serum containing both liposomal and free doxorubicin through SPE cartridge after proper conditioning and equilibration. Free doxorubicin gets retained on SPE cartridge with reversed phase interaction. Whereas liposomal doxorubicin will be passed on through the eluent. This collected eluent should be precipitated and can be quantified using a validated LC-MS/MS. This method was based upon the finding that liposomes can pass through solid-phase C18 silica gel cartridges without being absorbed, but free Doxorubicin is retained on the stationary phase.
Using ultracentrifugation based on molecular weight cutoff:
An alternate approach is passing plasma sample through 30KD molecular weight cutoff filter and upon centrifugation, only free doxorubicin (<30KD) gets passed through filter gets collected for further quantification. Liposomal doxorubicin (>30KD) present in the above cutoff filter can be used for further quantification with simple protein precipitation by disrupting liposomal formulation and required dilution using a validated LC-MS/MS method
Measurement of Total formulation in Tissues
Due to possibilities of rupture of lipid layers and leaking the encapsulated formulation while homogenizing the vital organs and tumors, it is possible to measure only total formulations in organs unlike it in plasma where separation is possible for encapsulated and non-encapsulated formulations.
Pharmacokinetics and Tissue Distribution of Liposomal formulations:
Human equivalent dose of Liposomal formulations injected intravenously through tail vein in Rats and Mice and blood/tissue samples are collected at pre-defined intervals to capture their pharmacokinetic/tissue distribution profiles.
Advantages of Liposomal Formulations
Liposomal formulations have shown the advantages of longer half-life with high circulating blood concentrations and minimal distribution into the untargeted tissues resulting in minimal toxicity in comparison with non-liposomal formulations of the same drug.
Dabur Research Foundation:
At Dabur Research Foundation, we are supporting all our sponsors by conducting Pharmacokinetic and Tissue Distribution studies in Rats and Mice to know the newly prepared liposomal formulation kinetics before they enter clinical studies in patients. We have a state of art vivarium facility and bioanalytical laboratory with standard operating procedures to meet global standards. We are AAALAC accredited and GLP facility for conducting animal studies in non-clinical development.