Correlation of toxicity with pharmacokinetics of pegylated liposomal doxorubicin (Doxil) in metastatic breast carcinoma.

BACKGROUND: Doxil (ALZA Corp., Mountain View, CA) is a formulation of doxorubicin in polyethylene-glycol coated liposomes with a prolonged circulation time and unique toxicity profile. As yet, the effect of the dose schedule on toxicity and the correlation of toxicity with pharmacokinetics have not been directly addressed. METHODS: The objectives of this study were to examine the toxicity profile and pharmacokinetics of various dose schedules of Doxil in a group of patients with metastatic breast carcinoma (MBC) previously treated with chemotherapy. Forty-five patients received a total of 268 courses of Doxil (median per patient, 5; range, 1-19). Six dose schedules were investigated: 35 mg/m2 every 3 weeks (11 patients), 45 mg/m(2) every 3 weeks (5 patients), 50 mg/m(2) every 4 weeks (5 patients), 60 mg/m(2) every 4 weeks (6 patients), 65 mg/m(2) every 5 weeks (6 patients), and 70 mg/m(2) every 6 weeks (12 patients). Doxil pharmacokinetics was examined in 24 of these patients at the dose levels of 35, 45, 60, and 70 mg/m(2). RESULTS: Stomatitis was dose related, with higher incidence and severity at doses of 60-70 mg/m(2). Skin toxicity in the form of palmar-plantar erythrodysesthesia (PPE) developed usually after two or more courses of treatment and was schedule dependent with shorter dosing intervals leading to increased frequency and severity of skin manifestations. Myelosuppression, mainly as leukopenia/neutropenia, was dose dependent but mild and uncomplicated in most cases. Hair loss was infrequent (< 7%) and always of limited extent. Despite high cumulative doses up to 1500 mg/m(2), cardiac toxicity was observed in only 1 patient who received prior mitoxantrone and mediastinal radiotherapy. Objective responses, improvements, and durable stabilizations were observed in 9, 6, and 14 patients, respectively, indicating significant antitumor activity of Doxil in previously treated MBC patients. Doxil pharmacokinetics was well described by a monoexponential elimination curve with a long T(1/2) (median, 79 hours), a slow clearance (median, 40 mL/hour), and a small volume of distribution (median, 3.9 L). Cmax (peak plasma concentration) and AUC (area under the concentration*time curve) increased linearly with dose with a statistically significant correlation. Correlation analysis of dose and pharmacokinetic parameters with Doxil toxicites revealed that stomatitis grade and leukocyte nadir were correlated strongly with dose and Cmax, and weakly with AUC, whereas PPE grade was correlated significantly with only 1 parameter, T(1/2). CONCLUSIONS: The toxicity of Doxil is dose and schedule dependent and well correlated with pharmacokinetic parameters. Pharmacokinetic guidance of Doxil dosing may be a useful tool.

Nuclear delivery of doxorubicin via folate-targeted liposomes with bypass of multidrug-resistance efflux pump.

Folic acid, attached to polyethyleneglycol-derivatized, distearoyl-phosphatidylethanolamine, was used to target in vitro liposomes to folate receptor (FR)-overexpressing tumor cells. Confocal fluorescence microscopic observations demonstrated binding and subsequent internalization of rhodamine-labeled liposomes by a high FR-expressing, murine lung carcinoma line (M109-HiFR cells), with inhibition by free folic acid. Additional experiments tracking doxorubicin (DOX) fluorescence with DOX-loaded, folate-targeted liposomes (FTLs) indicate that liposomal DOX is rapidly internalized, released in the cytoplasmic compartment, and, shortly thereafter, detected in the nucleus, the entire process lasting 1-2 h. FR-mediated cell uptake of targeted liposomal DOX into a multidrug-resistant subline of M109-HiFR cells (M109R-HiFR) was unaffected by P-glycoprotein-mediated drug efflux, in sharp contrast to uptake of free DOX, based on verapamil-blockade experiments with quantitation of cell-associated DOX and flow cytometry analysis. Delivery of DOX by FTLs to M109R-HiFR cells increased continuously with time of exposure, reaching higher drug concentrations in whole cells and nuclei compared with exposure to free DOX. The in vitro cytotoxic activity obtained with DOX-loaded FTLs was 10-fold greater than that of the nontargeted liposome formulation, but was not improved over that of free DOX despite the higher cellular drug levels obtained with the targeted liposomes in M109R-HiFR cells. However, if M109R-HiFR cells were exposed to drugs in vitro and tested in an in vivo adoptive assay for tumor growth in syngeneic mice along a 5-week time span, FTL DOX was significantly more tumor inhibitory than free DOX. It is suggested that the biological activity of liposomal DOX released inside the cellular compartment is reduced in vitro due to the aggregated state of DOX, resulting from the liposome drug-loading process, and requires a long period of time and/or an in vivo environment for full expression.

Selective delivery of doxorubicin to patients with breast carcinoma metastases by stealth liposomes.

BACKGROUND: Stealth liposomes hold promise as a mode of delivering cytotoxic agents selectively to tumors in cancer patients. The objective of this study was to determine whether stealth liposomal doxorubicin accumulates selectively in bone metastases based on clinical material obtained from two patients with breast carcinoma. METHODS: Tumor tissue was obtained from two women (ages 33 years and 41 years, respectively) with metastatic breast carcinoma who responded to treatment with stealth liposomal doxorubicin and later underwent a surgical fixation procedure to treat a pathologic fracture of the femur. Drug levels in the tumor and adjacent muscle were examined by high performance liquid chromatography analysis in both patients and by fluorescence microscopy in one of the patients. RESULTS: Bone tumor fragments obtained during surgery performed 6 days after the administration of the 12th course of stealth liposomal doxorubicin in 1 patient and 12 days after the administration of the 16th course of stealth liposomal doxorubicin in the second patient had a 10-fold greater concentration of liposomal doxorubicin than tumor free muscle. Doxorubicin fluorescence and specific nuclear staining showed good colocalization, thus confirming the presence of the liposome-delivered drug in the nuclei of tumor cells. CONCLUSIONS: Using skeletal muscle as a comparator, stealth liposomal doxorubicin accumulates selectively in metastatic breast carcinoma cells within bone.

Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes: in vitro studies.

Conjugates of three components, folic acid-poly(ethylene glycol)-distearoylphosphatidylethanolamine (FA-PEG-DSPE), derived from PEG with molecular masses of 2000 and 3350 Da were synthesized by a carbodiimide-mediated coupling of FA to H2N-PEG-DSPE. The conjugates were characterized by 1H NMR, MALDI-TOF, and HPLC analysis of enzymatic cleavage with carboxypeptidase G. As a prototype of a folate receptor (FR)-targeted system, the conjugates were formulated at 0.5 mol % phospholipid in hydrogenated phosphatidylcholine/cholesterol liposomes with or without additional methoxyPEG2000-DSPE. In vitro binding studies were performed with sublines of M109 (murine lung carcinoma) and KB (human epidermal carcinoma) cells each containing high and low densities of FR. FA-PEG-DSPE significantly enhanced liposome binding to tumor cells. The best binding was observed when FA-PEG liposomes contained no additional mPEG-lipid. In fact, our experiments showed that the presence of mPEG on liposomal surfaces significantly inhibited FA-PEG-liposome binding to FR. Increasing the molecular mass of the PEG tether from 2000 to 3350 Da improved the FR binding, particularly in the case of mPEG-coated liposomes. The FA-PEG liposomes bound to M109-HiFR cells very avidly as demonstrated by the inability of free FA (used in a 700-fold excess either at the beginning or at the end of the incubation) to prevent the cell binding. This is in contrast to the 5-10-fold lower cell binding activity of mPEG-FA compared to that of free FA, and likely to be related to the multivalent nature of the liposome-bound FA. Only 22% of FA-PEG3350 and 32% of FA-PEG3350/mPEG cell-associated liposomes could be removed by exposure to pH 3, conditions that dissociate FA-FR, suggesting that more than two-thirds of the bound liposomes were internalized during incubation for 24 h at 37 degrees C. FA-targeted liposomes also show enhanced nonspecific binding to extracellular tissue culture components, a phenomenon especially relevant in short incubation time experiments.

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.

Comparative study of the antitumor activity of free doxorubicin and polyethylene glycol-coated liposomal doxorubicin in a mouse lymphoma model.

Here, we investigate various factors affecting the therapeutic efficacy of free doxorubicin (Free-Dox) and polyethylene glycol (PEG)-coated (PEGylated) liposomal doxorubicin (referred to as Doxil) in the ascitic J6456 lymphoma model of BALB/c mice. Free drug and liposomal drug were affected differently by the tumor burden and route of treatment administration. A delay in start of treatment from day 1 to day 5 almost completely abolished the efficacy of Free-Dox, whereas that of Doxil was only minimally reduced. Contrasting effects on the therapeutic efficacy of Free-Dox and Doxil were obtained by changing treatment administration from the i.v. to the i.p. route; the efficacy of free drug was relatively enhanced, whereas that of liposomal drug was relatively diminished. Overall, Doxil given by the systemic i.v. route was the most effective treatment in prolonging median survival and obtaining cures. Variations in the dose-schedule treatment regime confirm the superior therapeutic profile and reduced dependence on tumor burden of the PEGylated liposomal formulation over free drug. In addition, these experiments indicate that, at equal dose intensity, the dose level is more important than the frequency of administration for therapeutic activity.

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.