Purpose: Pharmacokinetic studies on liposomal drugs have previously measured total drug levels in tumors, which include nonbioavailable drug. However, drugs must be released from liposomes to have activity. We have developed a method for measuring levels of bioavailable (released) doxorubicin in vivo in tumors that will allow therapeutic activity to be correlated with bioavailable drug levels. Experimental Design: Mice orthotopically implanted with mammary carcinoma (4T1) were injected i.v. 10 days after implantation with free doxorubicin or formulations of liposomal doxorubicin with different drug release rates. Tumors were excised at various times after injection, and total tumor doxorubicin levels were determined by acidified isopropanol extraction of whole tumor homogenates. Bioavailable doxorubicin levels were determined by extraction of doxorubicin from isolated tumor nuclei. Results: Free doxorubicin had high levels of bioavailability in tumor tissue; 95% of the total doxorubicin in tumors was bound to nuclear DNA by 24 hours after injection. Administration of Doxil, a slow release liposomal formulation of doxorubicin, gave an area under the time-versusconcentration curve (AUC) for total doxorubicin 7 days after injection that was 87-fold higher than that obtained for free doxorubicin, and 49% of the liposomal doxorubicin was bioavailable. For liposomes with a more rapid doxorubicin release rate, by 7 days after injection, the AUC 0-7 days for total doxorubicin was only 14-fold higher than that for free doxorubicin and only 27% of liposomal doxorubicin was bioavailable. Conclusions: This technique allows correlations to be made between drug bioavailability and therapeutic activity and will help in the rational design of drug carriers.Long-circulating pegylated liposomal formulations of doxorubicin, Doxil, have been shown to result in increased accumulation of drug in solid tumors and reduced dose-limiting toxicities such as myelosuppression and cardiotoxicity. This is due to alterations in the pharmacokinetics and biodistribution of the encapsulated drug (1 -3). Doxil is currently approved for use in AIDS-related Kaposi sarcoma, refractory ovarian cancer, and metastatic breast cancer (4 -8).Doxorubicin-loaded liposomes have enhanced efficacy in some solid tumors compared with free doxorubicin, because they passively target solid tumors through the enhanced permeability and retention effect (9, 10), resulting in increased drug payloads delivered to tumors. The enhanced permeability and retention effect is a result of defective vascular endothelial linings of growing tumors, resulting in gaps in the endothelium up to f800 nm in diameter, which are large enough to permit the extravasation of liposomes with diameters in the range of 100 nm (11). In addition, growing tumors have defective lymphatic drainage, which contributes to the extended residence time of extravasated liposomes in the interstitial space of the tumor. Liposomes residing in the interstitial space gradually release their entrapped dr...