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dependent ductal adenocarcinoma that over-expresses C-ErbB2 (HER2) receptor. BT-474 cells were obtained from American Type Culture Collection (Rockville, Md.). A BT-474 sub-line with higher tumor growth rate was estab› lished from a fast-growing xenograft tumor nodule raised as described below. The cells were propagated in vitro in RPMI-1460 medium with 10% fetal calf serum, 0.1 mg/mL streptomycin sulfate, and 100 U/ml Penicillin Gin T-150 flasks, and split 1 :3 every week. NCR nu/nu female mice ( 4-6 week old; Taconic Farms) were subcutaneously implanted (at the base of tail) with 60-day sustained-release 0.72-mg 17~-estradiol pellets (Innovative Research of America, Inc.), and in two days were inoculated subcutane› ously in the upper back area with 0.1 mL suspension containing 2xl0 7 BT-474 cells in cell growth medium. The tumor progression was monitored by palpation and caliper measurements of the tumors along the largest (length) and smallest (width) axis twice a week. The tumor sizes were determined twice weekly from the caliper measurements using the formula (Geran, R. I., et al., 1972 Cancer Chemother. Rep. 3:1-88): 

<img src='content_image/938293.jpg'>

At day 13 after inoculation, the tumor reached an average size of 200 mm $^{3 }$and the animals were randomly assigned to three groups of 13-15 animals. 

Liposomal CPT-11 was prepared as in Example 8 ( drug/ phospholipid ratio 192 mg/mmol; average liposome size 86.8 nm). Free and liposomal CPT-11 were diluted with MES-dextrose vehicle to 5 mg/ml of CPT-11 base The animals were injected via the tail vein with free CPT-11, liposomal CPT-11, or vehicle only on days 14, 18, 21, and 25 post tumor inoculation. The drug-containing formula› tions were given at the dose of 50 mg CPT-11/kg per injection, which is the average of the doses reported in the literature for the CPT-11 studies in rodent tumor models. 

To assess treatment-related toxicity, the animals were also weighted twice weekly. The observations were made until day 60 post inoculation, at which time the estrogen supple› mentation pellet was exhausted. Average tumor volumes across the groups were plotted together and compared over time. As shown in FIG. 3, while free CPT-11 reduced the rate of tumor growth, in the group that received liposomal treatment the tumors regressed dramatically. While at day 36 in the control group the tumors reached the maximum allowable size averaging 3,500 mm 3 , and at day 46 in the group treated with free drug the tumors were about 1,000 mm 3 at average, at the same time point none of the animals in the liposome-treated group had a palpable tumor. 

The treatment related toxicity was assessed by the dynam› ics of animals’ body weight (FIG. 4). Neither group revealed any significant toxicity. The weight of the animals in the control group was consistently increasing. There was a slight decrease in the average body weight of the animals receiving liposomal CPT-11, by about 3.3%, on the day of the last treatment. This weight loss was reversed, however, and the animals reached their expected weight. This decrease in the mean body weight was not statistically significant by Stu› dent’s t-test compared to pretreatment weight (p=0.274). Thus, all treatments were tolerated without significant tox› icity. 

Thus, the liposome formulation of CPT-11 obtained by loading of the drug via pre-entrapped sterically hindered substituted ammonium salt (triethylammonium) of a poly- anionic, biodegradable polymer (polyphosphate) showed extended blood life, sustained release characteristics, and 

increased antitumor activity in the studied tumor model without an appreciable increase in toxicity. 

## Comparative Assessment of CPT-11 Loaded Liposomes Prepared Using Pre-Entrapped Triethylammonium Salts: Effect of Liposome Size, Drug/Lipid Ratio, and the Nature of Pre-Entrapped Anion 

Two prototype formulations of CPT-11-loaded liposomes were prepared, one using the liposomes with pre-entrapped TEA-Pn, and the other with pre-entrapped TEA-SOS. Prepa› ration of these liposomes included the following manufac› turing steps. 

1) Combining The Lipids By Co-Dissolving In Ethanol. The lipid matrix composition consisted of 1,2-Distearoyl› SN-phosphatidylcholine (DSPC) (Mo!. wt. 790) 3 molar parts (59.8 mo!.%); Cholesterol (Chol) (Mo!. weight 387) 2 molar parts (39.9 mo!. %); and N-(omega-methoxy-poly ( ethylene glycol)-oxycarbonyl)-1,2-distearoylphosphatidyl ethanolamine (Mo!. weight 2787) (PEG-DSPE) 0.015 molar parts (approx. 0.3 mo!. %). DSPC and PEG-DSPE were purchased from Avanti Polar Lipids, Birmingham, Ala. Cholesterol (highest purity grade) was purchased from Cal› biochem. Dry lipids were weighed with the accuracy of +0.1 mg in a borosilicate glass container and combined with absolute ethanol at the ratio suitable for the lipid dispersion step below. Because of the high transition temperature of DSPC (55° C.) the dissolution was typically performed at 55-60° C. until clear solution was obtained. 

2) Preparing the TEA-Pn and TEA-SOS Solutions. Sodium polyphosphate (n=13-18) was from Sigma Chemi- cal Co., p/n P 8510. Sodium sucrose octasulfate was pur› chased from Toronto Research Chemicals, Toronto, Canada, p/n S699020. The salts were weighed down and dissolved in water to provide 1.2-2.5 N solutions. Anion exchangers Dowex 50Wx8-200 or Dowex HCR-W2 in H+-form (avail- able from Sigma) were used to convert the sodium salts into free acids. Before the first use, the resins were washed by stirring with 3 volumes of the following solutions, followed by decanting: (1) 1.0-1.2 M aqueous HCl 2 times; (2) Water 2 times; (3) 1.0-1.2 M aqueous NaOH 2 times; (4) Water 2 times; (5) 1.0-1.2 M aqueous HCL, 2 times. The suspension of washed resin in water was packed under gravity flow in a suitable size chromatography column to have at least 8 mL of the packed resin for each mL of the sodium salt solutions. The resin was further equilibrated by passage of 2 column volumes of 3.0-3.6 M aqueous HCL, followed by 5 column volumes of water or until the conductivity of the eluate falls below lmicro-S. After use, the colunms were regenerated by sequential passage of: 1.0-1.2 M HCl-3 column volumes; 3.0-3.6 M HCl-2 colunm volumes; water-at least 5 column volumes, or until the conductivity of the eluate falls below 1 µS, and stored under 0.2-um filtered water at room temperature. The Pn and SOS sodium salt solutions were applied on the drained surface of the column (approximately 1 ml for each 4 ml of the packed resin volume) and allowed to flow under gravity at the rate of about 1-2 ml/min for the resin bed size of 75-150 mL. The column was eluted with water. The eluate was tested for conductivity. The fractions with 10 mS or higher conductivity were collected. If more concentrated solutions of polyacids are required, the collec› tion can start at 20-50 mS, but at the expense of somewhat higher loss of the gradient-forming salt. In the case of polyphosphoric acid, the collected solution is kept refriger- 

## Example 11 

