Reduced toxicity and superior therapeutic activity of a mitomycin C lipid-based prodrug incorporated in pegylated liposomes.

PURPOSE: A lipid-based prodrug of mitomycin C [MMC; 2,3-(distearoyloxy)propane-1-dithio-4'-benzyloxycarbonyl-MMC] was designed for liposome formulation. The purpose of this study was to examine the in vitro cytotoxicity, pharmacokinetics, in vivo toxicity, and in vivo antitumor activity of this new lipid-based prodrug formulated in polyethylene glycol-coated (pegylated) liposomes. EXPERIMENTAL DESIGN: MMC was released from the MMC lipid-based prodrug (MLP) by thiolytic-induced cleavage with a variety of thiol-containing reducing agents. MLP was incorporated with nearly 100% efficiency in cholesterol-free pegylated liposomes with hydrogenated phosphatidylcholine as the main component and a mean vesicle size of approximately 90 nm. This formulation was used for in vitro and in vivo tests in rodents. RESULTS: In vitro, the cytotoxic activity of pegylated liposomal MLP (PL-MLP) was drastically reduced compared with free MMC. However, in the presence of reducing agents, such as cysteine or N-acetyl-cysteine, its activity increased to nearly comparable levels to those of free MMC. Intravenous administration of PL-MLP in rats resulted in a slow clearance indicating stable prodrug retention in liposomes and long circulation time kinetics, with a pharmacokinetic profile substantially different from that of free MMC. In vivo, PL-MLP was approximately 3-fold less toxic than free MMC. The therapeutic index and absolute antitumor efficacy of PL-MLP were superior to that of free MMC in the three tumor models tested. In addition, PL-MLP was significantly more active than a formulation of doxorubicin in pegylated liposomes (DOXIL) in the M109R tumor model, a mouse tumor cell line with a multidrug-resistant phenotype. CONCLUSIONS: Delivery of MLP in pegylated liposomes is a potential approach for effective treatment of multidrug-resistant tumors while significantly buffering the toxicity of MMC.

Nuclear localization signal-targeted poly(ethylene glycol) conjugates as potential carriers and nuclear localizing agents for carboplatin analogues.

Carboplatin is a low-molecular-weight anticancer drug that acts by binding to the nuclear DNA of cells. Thus, efficient delivery of the platinum drugs to the nucleus of the cancer cells may enhance the cytotoxicity of the drug. Efficient drug delivery to the nucleus of cancer cells requires three levels of localization: targeting to the cancerous tissue, accumulation in the cancer cells, and intracellular localization in the nucleus. Nuclear localization signals (NLS) are short positively charged basic peptides that actively transport large proteins across the nuclear membrane. We have prepared conjugates in which the NLS is tethered to poly(ethyleneglycol)carboplatin conjugate (NLS-PEG-Pt) and compared their pharmacological properties to those of their untargeted analogues that do not possess the NLS (PEG-Pt). NLS-PEG-Pt conjugates are rapidly internalized into cancer cells and accumulate in the nucleus. Despite their rapid nuclear localization, they form less Pt-DNA adducts than the untargeted analogues, PEG-Pt, and are also less cytotoxic. These results support the hypothesis that carboplatin (unlike cisplatin) may require cytosolic activation prior to its binding to nuclear DNA.

Tumor cell targeting of liposome-entrapped drugs with phospholipid-anchored folic acid-PEG conjugates.

Targeting of liposomes with phospholipid-anchored folate conjugates is an attractive approach to deliver chemotherapeutic agents to folate receptor (FR) expressing tumors. The use of polyethylene glycol (PEG)-coated liposomes with folate attached to the outer end of a small fraction of phospholipid-anchored PEG molecules appears to be the most appropriate way to combine long-circulating properties critical for liposome deposition in tumors and binding of liposomes to FR on tumor cells. Although a number of important formulation parameters remain to be optimized, there are indications, at least in one ascitic tumor model, that folate targeting shifts intra-tumor distribution of liposomes to the cellular compartment. In vitro, folate targeting enhances the cytotoxicity of liposomal drugs against FR-expressing tumor cells. In vivo, the therapeutic data are still fragmentary and appear to be formulation- and tumor model-dependent. Further studies are required to determine whether folate targeting can confer a clear advantage in efficacy and/or toxicity to liposomal drugs.

In vivo fate of folate-targeted polyethylene-glycol liposomes in tumor-bearing mice.

PURPOSE: To compare the in vivo tissue distribution of folate-targeted liposomes (FTLs) injected i.v. in mice bearing folate receptor (FR)-overexpressing tumors (mouse M109 and human KB carcinomas, and mouse J6456 lymphoma) to that of nontargeted liposomes (NTLs) of similar composition. EXPERIMENTAL DESIGN: A small fraction of a folate-polyethylene-glycol (PEG)-distearoyl-phosphatidylethanolamine conjugate was incorporated in FTLs. Both FTLs and NTLs were PEGylated with a PEG-distearoyl-phosphatidylethanolamine conjugate to prolong circulation time. Liposomes were labeled with [(3)H]cholesterol hexadecyl ether with or without doxorubicin loading. Liposome levels in plasma, tissues, or ascites were assessed by the number of [(3)H] counts. For doxorubicin-loaded formulations, we also determined the tissue doxorubicin levels by fluorimetry. To estimate the amount of liposomes directly associated with tumor cells in vivo, we determined the [(3)H]radiolabel counts in washed pellets of ascitic tumor cells using the ascitic J6456 lymphoma RESULTS: FTLs retained the folate ligand in vivo, as demonstrated by their ability to bind ex vivo to FR-expressing cells after prolonged circulation and extravasation into malignant ascitic fluid. In comparison with NTLs, FTLs were cleared faster from circulation as a result of greater liver uptake. Despite the lower plasma levels, tumor levels of FTL-injected mice were not significantly different from those of NTL-injected mice. When NTLs and FTLs were loaded with doxorubicin, liver uptake decreased because of liver blockade, and uptake by spleen and tumor increased. When tumor-to-tissue liposome uptake ratios were analyzed, the targeting profile of FTLs was characterized by higher tumor:skin, and tumor:kidney ratios but lower tumor:liver ratio than NTLs. After a concomitant dose of free folic acid, FTLs (but not NTLs) plasma clearance and liver uptake were inhibited, indicating that accelerated clearance was mediated by the folate ligand. Surprisingly tumor uptake was not significantly affected by a codose of folic acid. In the J6456 ascitic tumor model, tumor cell-associated liposome levels were significantly greater for FTL-injected mice than for NTL-injected mice, despite slightly higher levels of the latter in whole ascites. CONCLUSIONS: Whereas folate targeting does not enhance overall liposome deposition in tumors, the targeting profile of tumor versus other tissues is substantially different and intratumor liposome distribution in ascitic tumors is affected favorably with a selective shift toward liposome association with FR-expressing cells.

Folate-targeted PEG as a potential carrier for carboplatin analogs. Synthesis and in vitro studies.

Like most low molecular weight drugs, carboplatin has a short blood circulation time, which reduces tumor uptake and intracellular DNA binding. Drugs conjugated to PEG carriers benefit from prolonged blood circulation, but suffer from reduced cell permeability. In this work we attempted to develop long-circulating PEGylated carboplatin analogues with improved cell permeation abilities, by conjugating the platinum moiety to folate-targeted PEG carriers capable of utilizing the folate receptor-mediated endocytosis (FRME). Two bifunctional FA-PEG conjugates, FA-PEG-Pt and FA-PEG-FITC, were prepared, and their cell uptake, DNA binding, and cytotoxicity were studied by fluorescent microscopy, FACS, and platinum analysis. Folate-targeted PEG conjugates enter the cells efficiently by the FRME pathway but form relatively few DNA adducts and have higher IC(50) values than carboplatin and their nontargeted analogues. Nontargeted PEG-Pt conjugates have a lower cellular uptake but produce higher levels of DNA binding and improved cytotoxicity. Carboplatin, used as a control, has the fastest cellular uptake, but after 16 h of postincubation a large percentage of the drug is excreted from the cells. The findings of this study suggest that folate-targeted conjugates such as FA-PEG-Pt, may not be an optimal prodrug for the carboplatin family compounds, because the conjugates or the active moieties are neutralized or blocked during the FRME process and do not manage to effectively reach the nuclear DNA.

Dose dependency of pharmacokinetics and therapeutic efficacy of pegylated liposomal doxorubicin (DOXIL) in murine models.

Stealth (pegylated) liposomal doxorubicin (Doxil) has been extensively studied at the pre-clinical and clinical level in recent years. However, one issue not yet addressed is the effect of dose on tumor localization and therapeutic efficacy of Doxil. Although it has been reported that the pharmacokinetics of drug-free Stealth liposomes is independent of dose within a certain range, clinical pharmacokinetic analysis of Doxil suggests a dose-dependent clearance saturation phenomenon when a broad dose range is examined. In addition, liposome-encapsulated doxorubicin can exert toxic effects on the liver macrophage population in the form of impairment of the phagocytic function and reduced ability of colloid particle clearance. In studies with tumor-bearing mice in which the dose of Doxil was escalated from 2.5 to 20 mg/kg, we demonstrate that dose escalation results in a saturation of Doxil clearance and a disproportional increase of the amount of liposomal drug accumulating in tumor. Experiments with radiolabeled highly negatively-charged liposomes injected into mice previously treated with Doxil are consistent with a partial blockade of the reticulo-endothelial system with relative reduction of liver uptake and greater prolongation of liposome circulation time. The clearance saturation effect is seen after Doxil in a dose-dependent fashion, and not after a similar free doxorubicin dose or similar phospholipid dose in drug-free liposomes. A trend to superior therapeutic efficacy for treatments based on larger doses as compared to smaller split doses, while maintaining an equivalent dose intensity, was also observed. These observations may be relevant to the choice of dose-schedule of Doxil to ensure optimal anti-tumor activity. Therefore, dose-dependent liposomal doxorubicin blockade of the reticulo-endothelial system may prolong liposome circulation time and enhance significantly drug delivery to tumors.