Aviva T. Horowitz

Long-circulating liposomes for drug delivery in cancer therapy: a review of biodistribution studies in tumor-bearing animals

Inhibition of the rapid uptake of liposomes by the reticulo-endothelial system (RES) and reduction of the rate of drug leakage have resulted in long-circulating liposomal drug systems with valuable pharmacologic properties. Particularly, the coating of liposomes with polyethylene-glycol (PEG) confers optimal protection to the vesicles from RES-mediated clearance, while bilayer rigidification using high Tm phospholipids reduces the rate of leakage of liposome contents. These carrier systems display an improved extravasation profile with enhanced localization in tumors and possibly in other tissues, such as skin. An anti-cancer drug, doxorubicin, encapsulated in small-sized (< 100nm diameter), PEG-coated liposomes with a rigid bilayer shows a unique pharmacokinetic pattern, characterized by extremely long half-life, slow clearance, and small volume of distribution. Liposome longevity in circulation correlates positively with high drug levels in the extracellular tumor fluid and with enhanced therapeutic efficacy in a variety of tumor models regardless of the site of tumor growth. Examples of biodistribution studies will be presented for several murine tumors and human tumor xenografts inoculated by various routes, including a brain-implanted tumor. Liposome localization in tumors appears to be the result of an enhanced rate of extravasation through the abnormally permeable microvasculature of tumors coupled with an impaired lymphatic drainage. These results stress the potential of these long-circulating liposomal systems to manipulate the pharmacokinetics of anticancer drugs and enhance drug delivery to tumors. This therapeutic approach has been validated in AIDS-related Kaposis sarcoma and is now undergoing extensive clinical testing in solid tumors.

Pharmacological studies of cisplatin encapsulated in long-circulating liposomes in mouse tumor models.

We investigated the pharmacokinetics and therapeutic efficacy of cisplatin encapsulated in polyethyleneglycol-coated long-circulating liposomes in a formulation referred to as SPI-077, in three mouse tumor models (M-109 lung carcinoma inoculated s.c., J-6456 lymphoma inoculated i.p. and A-375 melanoma inoculated s.c.). Tumor-bearing mice were injected i.v. with single doses of SPI-077 and cisplatin. For pharmacokinetic experiments, mice were sacrificed at different timepoints post-treatment. Platinum levels were determined in plasma, spleen, liver, kidneys and tumors using flameless atomic absorption spectrophotometry. Survival times and/or tumor size were recorded for therapeutic studies. The pharmacokinetic studies revealed a prolonged circulation time and enhanced tumor uptake for SPI-077. In contrast to these results, no superior antitumor activity of SPI-077 over cisplatin could be observed in all tumor models. In vitro release experiments showed a negligible release (below 10%) of platinum from the liposomes. An in vitro cytotoxicity assay indicated a reduced cytotoxic activity of SPI-077 in comparison to cisplatin. We concluded that SPI-077 is being delivered to the tumor sites in a low bioavailability form, with extremely slow release kinetics. This explains the discrepant results of high platinum concentrations in tumors and reduced therapeutic activity after administration of SPI-077.

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.

Liposome Longevity and Stability in Circulation: Effects on the in vivo Delivery to Tumors and Therapeutic Efficacy of Encapsulated Anthracyclines

The effect of liposome composition on drug delivery to tumors and therapeutic efficacy of liposome-encapsulated anthracyclines was investigated in two murine tumor models: an ascitic tumor (J6456 lymphoma) and a solid carcinoma (M-109). Longevity in circulation correlated positively with high drug levels in the extracellular (ascitic) tumor fluid and with delayed peak tumor levels. Using polyethylene-glycol(PEG)-coated liposomes, liposome stability (drug retention) was found to be an important determinant of therapeutic efficacy, as indicated by the superior survival conferred by high Tm phosphatidylcholines (hydrogenated, dipalmitoyl) over low Tm (egg phosphatidyl-choline). Replacing PEG with another negatively-charged surface headgroup (phosphatidyl-glycerol, phosphatidyl-inositol) resulted in relatively shorter longevity in circulation of the liposome-associated drug, but no detectable differences in anti-tumor efficacy. When neither the surface charged headgroup nor the PEG coating are present, the resulting drug formulation was significantly less effective than PEG and phosphatidylinositol-based formulations in both tumor models. In conclusion, longevity in circulation, as obtained with PEG coating, tends to improve the therapeutic efficacy of liposome-encapsulated anthracyclines. The current therapeutic models were however unable to detect differences between the therapeutic activity of PEG and other liposome formulations with relatively small differences in circulation longevity.

The Mechanism of Human Bladder Tumor Implantation in an in Vitro Model

Implantation of tumor cells in the bladder following transurethral resection of superficial bladder tumors is believed to be one factor in the etiology of bladder tumor recurrences. Using an in vitro model system we have studied the initial interaction between bladder carcinoma cells and a naturally produced basement membrane-like substrate. Minced explants of superficial low grade human bladder tumors from 10 patients were plated into culture dishes coated with a naturally produced extracellular matrix (ECM). This ECM has been shown to resemble the human urothelial basement membrane and submucosa in its macromolecular composition and ultrastructural appearance. It was found that a firm attachment of the human bladder tumor cells occurred within one hour, reached a maximal value within 24 hours and was followed by flattening and proliferation of the plated cells. These results indicate that prevention of tumor implantation should be initiated in the first hour after transurethral resection of the bladder tumors. This assay can be used for the investigation of various treatments to prevent tumor implantation.

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 ∼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 ∼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.

A New Human Ovarian Carcinoma Cell Line: Establishment and Analysis of Tumor-Associated Markers

In the present study we describe the establishment and characteristics of a new human tumor cell line (OV-1063) positive for carcinoembryonic antigen (CEA) originating from ovarian metastatic tumor cells. Analysis of the cultured cells during their in vitro adaptation period revealed while the primary culture exhibited a low proportion of CEA-positive cells, this proportion increased with culture passages and eventually more than 90% of the cells in the established line were CEA-positive. Thus, during the period of adaptation to in vitro growth, a selection for CEA-positive cells took place but the amount of CEA secreted per each positive cell seemed to be constant. Several tumor-associated characteristics were found positive on the established OV-1063 cell line. The in vitro growing cell line exhibited an abnormal chromosome pattern with a near-trisomy karyotype for some chromosomes, colony formation in soft agar as well as positive staining with a monoclonal antibody B38.1. Culture supernatants of the OV-1063 cells contained significant amounts of CEA as well as CA-125 antigen which is an ovarian-carcinoma-associated antigen.

Preclinical Studies with Doxorubicin Encapsulated in Polyethyleneglycol-Coated Liposomes

AbstractDoxorubicin (DOX) has been encapsulated with high efficiency in the water phase of small-sized lipid vesicles. Plasma-induced drug leakage from these vesicles is minimal when hydrogenated phosphatidylcholine is present as the main component. A prolonged circulation time of liposome-encapsulated DOX is observed in animal models when a small fraction of polyethyleneglycol-derivatized phospholipid (PEG) is present in the liposome bilayer. Using these PEG-coated liposomes, we found that the concentration of DOX in tumor implants of the mouse M-109 carcinoma is significantly enhanced by liposome delivery. The antitumor activity of liposome-encapsulated DOX in a lung metastases model of the M-109 carcinoma is superior to that of free DOX. The minimal lethal dose of DOX to tumor-free mice was substantially increased by encapsulation in PEG-coated liposomes, indicating that toxicity is reduced. We also found that the vesicant of DOX after intradermal injection is prevented by liposome encapsulation. These...

Prevention of Human Bladder Tumor Cell Implantation in an in Vitro Assay

The high recurrence rate of bladder tumors can be reduced by prevention of tumor cell reimplantation on denuded urothelium following transurethral resection. This can be achieved by intravesical chemotherapy immediately after the resection of the bladder tumors. We have demonstrated, in an in-vitro system, the process of human bladder tumor cell implantation on a naturally produced extracellular matrix (ECM) which simulates the exposed bladder basement membrane and submucosa. Using this model we examined the efficacy of various cytotoxic agents in preventing tumor cell adhesion to the ECM. Human bladder tumor cell implantation was prevented following exposure of the cells to distilled water, epodyl or mitomycin C, and significantly reduced following one hour incubation in cisplatinum and doxorubicin. The maximal effect for each of these cytotoxic agents was reached within 30 to 60 minutes of treatment. Mitomycin C reached maximal effect within 10 minutes. In contrast, thiotepa did not cause a significant reduction in cell adherence to ECM as compared to untreated control cells.

Stealth™ Liposomes as Carriers of Doxorubicin

Liposomes, as non-covalently bound carriers, biocompatible and biodegradable, have raised considerable interest as a drug delivery system in cancer chemotherapy (Gregoriadis, 1988). Most applications of liposomes in cancer chemotherapy are directed at altering tissue distribution and various pharmacokinetic parameters of the drug in question in such a way that toxicity can be reduced and/or efficacy increased (Mayhew and Papahadjopoulos, 1983). Reduced toxicity may be gained through site circumvention of drug sensitive tissues and by slow release of the cytotoxic agent from the carrier, avoiding peak plasma concentrations after bolus injection of free drug. Liposome- mediated decrease in toxicity could enable escalation of dose, which will result in increased tumor exposure to the drug.