Dorit Goren

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.

Targeting of stealth liposomes to erbB-2 (Her/2) receptor: in vitro and in vivo studies.

Long-circulating (stealth) liposomes coated with polyethylene glycol (PEG), which show reduced uptake by the reticuloendothelial system (RES) and enhanced accumulation in tumours, were used for conjugation to monoclonal antibodies (MAbs) as a drug-targeting device. A MAb (N-12A5) directed against erbB-2 oncoprotein, a functional surface antigen, was used. Amplification and overexpression of the erbB-2 gene product, being unique to malignancy, confer onto this antibody-mediated therapy high tumour specificity. In vitro binding of [3H]cholesteryl ether ([3H]Chol ether) labelled anti-erbB-2 conjugated liposomes to N-87 cells (erbB-2-positive human gastric carcinoma) was compared with the binding of non-targeted liposomes and indicated a 16-fold increase in binding for the targeted liposomes. No difference in binding to OV1063 cells (erbB-2-negative human ovary carcinoma) was observed. These results indicate highly selective binding of antibody-targeted liposomes to erbB-2-overexpressing cells. Despite increased cell binding, doxorubicin (DOX) loaded in anti-erbB-2-conjugated liposomes did not cause increased in vitro cytotoxicity against N-87 cells, suggesting lack of liposome internalisation. In vivo, the critical factor needed to decrease the non-specific RES uptake and prolong the circulation time of antibody-conjugated liposomes is a low protein to phospholipid ratio ( < 60 micrograms mumol-1). Using these optimised liposome preparations loaded with DOX and by monitoring the drug levels and the [3H]Chol ether label, biodistribution studies in nude mice bearing subcutaneous implants of N-87 tumours were carried out. No significant differences in liver and spleen uptake between antibody-conjugated and plain liposomes were observed. Nevertheless, there was no enhancement of tumour liposome levels over plain liposomes. Both liposome preparations considerably enhanced DOX concentration in the tumour compared with free drug administration. Therapeutic experiments with N-87 tumour-bearing nude mice indicated that anti-tumour activity of targeted and non-targeted liposomes was similar, although both preparations had an increased therapeutic efficacy compared with the free drug. These studies suggest that efficacy is dependent on drug delivery to the tumour and that the rate-limiting factor of liposome accumulation in tumours is the liposome extravasation process, irrespective of liposome affinity or targeting to tumour cells.

Development of liposomal anthracyclines : from basics to clinical applications

The pharmacokinetics of liposome-encapsulated drugs are controlled by the interplay of two variables: the rate of plasma clearance of the liposome carrier, and the stability of the liposome-drug association in the blood stream. The pharmacokinetic properties of the liposomal drug, the vesicle size of the liposome carrier and the vascular permeability of individual tissues will determine the extravasation and biodistribution profile. The pharmacokinetics of polyethylene-glycol-(PEG)-liposomal doxorubicin are characterized by an extremely long circulating half-life, slow plasma clearance and reduced volume of distribution compared to free doxorubicin. These carrier systems show an improved extravasation profile with enhanced localization in tumors and superior therapeutic efficacy in comparison to doxorubicin in free form. These properties are the result of an optimized liposome composition and of a special drug-loading method which produces stable and long-circulating carriers. In clinical studies, doxorubicin encapsulated in PEG-coated liposomes shows a unique pharmacokinetic-toxicity profile and promising antitumor activity.

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.

Superior therapeutic activity of liposome-associated adriamycin in a murine metastatic tumour model.

We have examined the anti-tumour activity of liposome-entrapped Adriamycin in a murine metastatic tumour model produced by i.v. inoculation of J-6456 lymphoma cells and affecting predominantly the liver. Sonicated liposomes containing phosphatidylcholine, a negatively-charged phospholipid and cholesterol were used in these experiments. Liposome-entrapped Adriamycin was more effective than free Adriamycin at equivalent doses of the drug. The superior therapeutic effect of the liposome-associated drug was manifest, either with a single i.v. treatment using a dose bordering the toxicity threshold of free Adriamycin or with a multi-injection schedule using smaller doses. Based on the growth kinetics data of the J-6456 lymphoma, our results indicate that tumour cell killing was enhanced by a factor of approximately 100 using the liposome associated form of Adriamycin. Histopathologic studies in mice bearing well-established metastases of the J-6456 lymphoma in liver and spleen indicated that the extent and duration of pathologic remission were significantly improved in mice receiving the liposome-entrapped drug as compared to mice receiving free drug. No significant differences in the anti-tumour effect of liposome entrapped Adriamycin were observed replacing phosphatidylserine by phosphatidylglycerol and reducing the cholesterol:phospholipid molar ratio from 100% to 25%. In contrast to the metastatic tumour model, liposome-entrapped Adriamycin was significantly less effective than free Adriamycin on the local i.m. growth of the J-6456 tumour. Altogether the survival and histopathological data presented suggest that, with regard to a group of neoplastic conditions with a predominant pattern of liver dissemination, a substantial increase in the therapeutic index of Adriamycin can be achieved in a selective manner with the use of liposomes.

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 influence of physical characteristics of liposomes containing doxorubicin on their pharmacological behavior

We have investigated the behavior of two populations of doxorubicin (DXR)-containing phospholipid vesicles with regard to various physical and pharmacological parameters. DXR-containing liposomes were prepared by ultrasonic irradiation, the lipid composition being phosphatidylglycerol (or phosphatidylserine), phosphatidylcholine and cholesterol. The vesicles were fractionated into oligolamellar vesicles (OLV) and small unilamellar vesicles (SUV) by preparative differential ultracentrifugation (150,000 x g for 1 h). Unentrapped DXR was removed by gel exclusion chromatography. OLV and SUV liposomes differed in size (mean diameters, 247 +/- 113 nm and 61 +/- 16 nm, respectively) and number of lamellae (two for OLV, one for SUV). Drug entrapment per unit of lipid was three to 5-fold higher in OLV than in SUV. In both liposome populations more than 95% of the entrapped drug was membrane-associated. Physical studies on these two vesicle populations revealed higher motional restriction and greater susceptibility to iodide-mediated fluorescence collisional quenching of DXR in the small vesicles. OLV showed superior stability in the presence of plasma as determined by the fraction of DXR retained by the vesicles. It was also found that the tissue distribution of DXR in SUV follows a pattern different from that of DXR in OLV and resembling that of soluble DXR. In accordance with these differences in patterns of tissue distribution, animal studies demonstrated that DXR in OLV is significantly less toxic than DXR in SUV and more effective in a tumor model with predominant involvement of the liver. These results indicate that vesicle size and/or number of lamellae play an important role in optimizing liposome-mediated delivery of DXR, and that oligolamellar liposomes are distinctively superior to small unilamellar liposomes when fluid phase formulations (Tm less than 37 degrees C) with bilayer-associated DXR are considered.

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...

Design, Characterization and Anti-Tumor Activity of Adriamycin-Containing Phospholipid Vesicles

In this report we describe the design of adriamycin (ADM)-containing liposome preparations aiming at optimization of various pre-established parameters. Regarding liposome composition phosphatidylserine (PS) and phosphatidylglycerol (PG) appear to be suitable negatively charged phospholipids which combined with phosphatidylcholine (PC) and cholesterol (CHOL), confer to liposomes high loading capacity for ADM and reasonable stability in plasma. Intravenous administration of these negatively-charged liposomes resulted in a favorable tissue distribution of ADM in both normal and tumor-bearing mice, characterized by decreased cardiac uptake of drug, and increased and sustained drug levels in the liver. Moreover, enhanced accumulation of drug also occurred in metastatic tumor cells isolated from the liver when ADM was injected in the liposome-associated form. This passive drug targeting resulted in an improved therapeutic efficiency of liposome-associated ADM in a tumor model of liver metastases. Liposome delivery of ADM was also shown to increase significantly its cytoreductive effect on spleen-infiltrating leukemia cells and to maintain the same cytoreductive efficiency on bone marrow residing leukemia cells with an overall favorable effect on survival in the BCL1 leukemia model. The reduction of ADM toxicity by liposome association together with the anti-tumor results indicate that liposomes represent a useful drug-delivery system for the treatment of major neoplastic conditions.

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.