Stable isotope method to measure drug release from nanomedicines

Abstract

Existing methods to measure nanomedicine drug release in biological matrices are inadequate. A novel drug release method utilizing a stable isotope tracer has been developed. Stable isotope-labeled drug is spiked into plasma containing nanomedicine. The labeled drug equilibrates with plasma components identical to the normoisotopic drug released from the nanomedicine formulation. Therefore, the ultrafilterable fraction of the isotope-labeled drug represents a reliable measure of free normoisotopic drug fraction in plasma, and can be used to calculate nanomedicine encapsulated and unencapsulated drug fractions. To demonstrate the utility of this method, we performed a plasma drug release study with both a fast releasing commercial docetaxel formulation, Taxotere®, and a delayed releasing nanomicellar formulation of a docetaxel prodrug, Procet 8. The instability of the unencapsulated prodrug in plasma allowed us to compare our calculated prodrug release and docetaxel conversion with the actual docetaxel concentration measured directly without fractionation. Drug release estimates for the fast releasing Taxotere formulation demonstrated accuracy deviation and precision (%CV) of <15%. For the controlled release Procet 8 formulation, we calculated a slow release and conversion of the prodrug in rat plasma that was highly correlated with the direct docetaxel measurement (R(2)=0.98). We believe that this method will have tremendous utility in the development and regulatory evaluation of nanomedicines, and aid in determination of generic bioequivalence.

Keywords: Drug release method; Fractionation in biological matrix; Nanomedicine; Nanotechnology; Stable isotope dilution.

Fig. 1

Stable isotope drug release method. In this method, the stable isotopically labeled drug (D*) equilibrates with protein (Pro) and formulation components identical to the unlabeled, normoisotopic drug (D) released from the nanomedicine (NM) formulation. Therefore, the ultrafilterable fraction of the isotopically labeled drug represents a reliable measurement of free drug fraction, and plasma protein bound fraction can be calculated from equation (i) in the text. The unencapsulated and encapsulated nanomedicine fractions can then be easily calculated, using equations (ii) and (iii) in the text, respectively.

Fig. 2

Protein binding comparison of docetaxel and docetaxel-d5 in human plasma. Protein binding of docetaxel (DTX) over a concentration range of 500-10,000 ng/mL, with 500 ng/mL deuterated docetaxel-d5 (DTX-d5) spike, was determined . Displayed are the unbound DTX concentrations, measured as the ultrafilterable drug concentrations, and the % bound drug for both DTX and spiked DTX-d5, calculated from equation (i) (mean ± SD, N=3).

Fig. 3

Correlation plot of theoretical versus method calculated docetaxel concentrations for protein binding comparison. Displayed are the individual theoretical versus calculated docetaxel (DTX) concentrations for each DTX concentration level.

Fig. 4

Drug release from Taxotere and solvent formulations in human plasma. Displayed are the % released docetaxel (DTX) for (A) Taxotere and (B) Acetonitrile solvent formulations, calculated from equation (ii) (mean ± SD, N=3).

Fig. 5

Drug release from Procet 8 in rat plasma. Displayed are the % released docetaxel (DTX) for each concentration and time point, calculated from equation (ii) (mean ± SD, N=3).

Fig. 6

Method control studies. Displayed are the released docetaxel (DTX) concentrations calculated from equation (ii), for a 10 μg/mL Procet 8 plasma sample, incubated for 6 h and centrifuged once or twice (spin 1 and spin 2, respectively), or spiked with 300 ng of docetaxel and centrifuged only once. The “spiked difference” was calculated as the difference between the spiked and non-spiked samples that were centrifuged only once (mean ± SD, N=3).

Fig. 7

Correlation plot of measured versus method calculated docetaxel concentrations. Displayed are the mean measured versus calculated DTX concentrations for all time points and concentrations (mean, N=3).