During early development of a nanomedicine, it is important to evaluate the formulation’s stability and drug release in biological matrices. In addition, quantifying the encapsulated and unencapsulated nanomedicine drug fractions is important for the determination of bioequivalence (pharmacokinetic equivalence) of generic nanomedicines (1). Since plasma protein binding for most marketed drugs is in excess of 70% (2) and can change in a concentration-, time-, and even formulation- dependent manner (3), accurate determination of the protein bound fraction is a considerable challenge. This assay utilizes an improved ultrafiltration method for nanomedicine fractionation in plasma, in which a stable isotope tracer is spiked into a nanomedicine containing plasma sample in order to precisely measure the degree of plasma protein binding (4) (Figure 1). Determination of protein binding then allows for accurate calculation of encapsulated and unencapsulated nanomedicine drug fractions, as well as free and protein-bound fractions.
This protocol is written to conduct an in vitro drug release study in human plasma, comparing drug release of a bilayer-loaded docetaxel (DTX) nanoliposome to the commercial DTX formulation, Taxotere®, and solvent solubilized DTX. Comparison data using this method were published previously (4). Additionally, the method can be applied to fractionate nanomedicine containing plasma from an in vivo pharmacokinetic study, such as for a bioequivalence trial. As long as a stable isotope labeled version of the free drug is available, the methods introduced here can be tailored to other nanomedicines. The stable isotope tracer is non-radioactive and generally a deuterated or carbon-13 isotope labeled analog of the normoisotopic drug encapsulated in the nanomedicine formulation. It is important that the isotope tracer is at least 3 amu different from the normoisotopic drug to ensure accurate mass separation and quantitation by mass spectroscopy.
- NCL_Method_PHA-2.pdf(PDF | 636 KB)
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