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...

Nanoscale drug delivery systems (DDS) are used to circumvent some of the non-ideal properties of conventional anticancer chemotherapy drugs. Manipulation of the physical properties of DDS provides improved control over the pharmacokinetics (PK) and pharmacodynamics (PD) of the encapsulated drugs relative to free drugs. Liposomes are the archetypical nanoscale DDS and the first of these received clinical approval in 1990. DOXIL, liposomal doxorubicin, was the first commercially available liposomal anticancer drug (1995). It has an enhanced circulation half-life compared to the free drug because of its surface-grafted polyethylene glycol coating. DOXIL passively targets solid tumors, and once the liposomes localize in the tumor interstitial space, the cytotoxic drug is slowly released within the tumor. Liposomes can act as sustained release delivery system and manipulation of properties such as, liposome diameter, drug release rate, bioavailability and dosing schedule can significantly impact the therapeutic outcome of the liposomal drugs. This review will focus on how alteration of these properties can impact the therapeutic efficacy and side effect profiles of DDS.

The selective toxicity of anticancer drugs can be improved with the use of antibody-targeted liposomes. We hypothesize that liposomes targeted via antibodies against two or more receptor populations will increase the apparent receptor density on the target cells, resulting in improved therapeutic affects. A fluorescent assay was developed, using the fluorophores Alexa Fluor 350 and 532 to label monoclonal antibodies (mAb), and used to quantitate two different mAb populations coupled to the same liposome surface to within +/-10% of the values obtained with radiolabeled antibody (125I) tracers. The binding and uptake of targeted liposomes by B lymphoma (Namalwa) cells were examined for either individual populations of alphaCD19-targeted or alphaCD20-targeted liposomes, mixed populations (1:1) of alphaCD19-targeted liposomes plus alphaCD20-targeted liposomes, and dual-targeted liposomes, i.e., equal amount of both alphaCD19 and alphaCD20 on the same liposomes. At similar antibody densities, the binding and uptake of the dual-targeted liposomes were greater than that of either individually targeted liposomes alone, and showed additivity. At the same total lipid and antibody densities, 1:1 mixtures of individually targeted liposomes gave similar results to dual-targeted liposomes. Cytotoxicity was also improved, with DXR-loaded dual-targeted liposomes appearing to have higher cytotoxicity than 1:1 mixtures of individually targeted liposomes.

Drugs that are entrapped in the interior of nanocarriers such as liposomes have no therapeutic activity, i.e. they are not bioavailable. In order to achieve therapeutic activity, drug release from the liposomes must occur at a rate sufficient to achieve therapeutic concentrations of drug at the cellular target. For ligand-targeted liposomes, directed against internalizing antigens, receptor-mediated internalization of the liposome package occurs and the entrapped drugs become active (bioavailable) upon their intracellular release from the lysosomal apparatus. We have examined, in a murine breast cancer model, the rate and the extent of bioavailability of doxorubicin (DXR) entrapped in liposomes targeted by a single-chain antibody fragment against the HER2/neu antigen, in comparison with free DXR and non-targeted liposomal DXR (DOXIL). Breast cancer tumors contained the highest total levels of DXR and the highest levels of bioavailable DXR when anti-HER2/neu-targeted liposomes were used, and the targeted liposomes also resulted in the greatest level of tumor control.