DETERMINATION OF FREE AND LIPOSOME-ASSOCIATED DOXORUBICIN AND VINCRISTINE LEVELS IN PLASMA UNDER EQUILIBRIUM CONDITIONS EMPLOYING ULTRAFILTRATION TECHNIQUES

A thorough understanding of the pharmacodynamic relationships associat ed with toxicity and efficacy behavior of liposome-encapsulated antica ncer agents such as doxorubicin and vincristine will rely on the abili ty to accurately separate and quantify the free and liposome-associate d drug fractions in plasma after administration. We have investigated the use of ultrafiltration as a method of isolating free doxorubicin a nd vincristine from liposomal drug under equilibrium conditions and co mpared it to previously developed nonequilibrium procedures based on s olid-phase extraction. Adsorption of drugs dissolved in saline to the ultrafiltration devices resulted in concentration-dependent ultrafiltr ate drug recoveries ranging from 41 to 96%. However, concentration-ind ependent quantitative recovery of vincristine in saline solutions coul d be obtained by passivating the ultrafiltration devices with PEG-8000 and device drug adsorption was ameliorated for both agents by plasma. The ultrafiltration method provided a more reliable separation of fre e and protein-bound drug, whereas solid-phase extraction yielded artif icially high free drug concentrations due to process-induced protein-b ound drug complex dissocation. Also, coelution of liposomes with the f ree drug fraction using solid-phase extraction was 64- to 418-fold hig her than observed with ultrafiltration, Taken together, these properti es indicated a significantly increased degree of accuracy in measuring the amount of free doxorubicin and vincristine in samples containing liposomal formulations employing ultrafiltration compared to solid-pha se extraction. The importance of this improvement was highlighted by o bservations that determinations of free drug concentrations in the pla sma of mice injected with liposomal doxorubicin and vincristine were 3 - to 12-fold higher using solid-phase extraction compared to ultrafilt ration. Finally, the ultrafiltra- tion procedure is rapid, versatile, and can be used for a wide range of drug and liposome concentrations a nd free drug/liposomal drug ratios. (C) 1995 Academic Press, Inc.