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):  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  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- 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.  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.  increased antitumor activity in the studied tumor model  without an appreciable increase in toxicity.  US 9,724,303 B2  38  37  Example 11  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  Tumor volume~[(length)x(width)2]/2 