US 9,717,723 B2  62  61  weight loss of about 15%. In the control group and free drug  group, there was no weight loss or treatment-related mor› bidity.  This study used a liposome Topotecan formulation pre› pared as in to Example 29, except the triethylammonium  polyphosphate solution was replaced with triethylammo› nium sucrose octasulfate solution having 0.65 M triethyl› ammonium, pH 6.2; and for extrusion 80-nm polycarbonate  membrane filters were used instead of 100-nm. Volume› weighted liposome size determined by quasi-elastic light  scattering method in Gaussian approximation (QELS) was  95.1–19.6  nm  (average–SD);  drug/lipid  ratio  was  369.1–18.3 mg/mmol phospholipid. Five-six week old  female Swiss-Webster mice (18-20 g) in the groups of two  received three i.v. (tail vein) injections of free or liposomal  Topotecan on a once-a-week schedule, starting from the  dose of 2 mg/kg Topotecan base per injection and increasing  to each subsequent group by the factor of 1.8 to the dose of  37.8 mg/kg. Immunoliposomal Topotecan was not included  in this study. Animal body weight and general health was  monitored daily. Progressive weight loss of more than 20%  or natural death at any time in any of two animals in a group  during the period of ten days since the beginning of treat- ment were considered indicative of the toxic dose. Accord› ing to the animal mortality and weight data MTD was  determined to fall within the range of 11.7-21 mg/kg for free  Topotecan, and 2.0-3.6 mg/kg for liposomal (Prototype 2)  Topotecan. In the second study, the mice received injections  of the free, liposomal, or F5-immunoliposomal Topotecan  (prepared from the liposomal Topotecan of this Example as  described in Example 29) with the doses from 2.0 mg/kg  (liposomal/immunoliposomal Topotecan) or 12 mg/kg (free  Topotecan), and increased to each subsequent group by the  factor of 1. 15 until the dose next to the upper range of the  established MTD interval was achieved. The highest dose  that did not result in death or terminal morbidity in any of  the animals was considered an MTD and was found to be  18.4 mg/kg for free Topotecan, 3.0 mg/kg for liposomal  Topotecan, and 3.0 mg/kg for immunoliposomal Topotecan.  Thus, liposomal Topotecan showed greater toxicity than the  free drug.  The Topotecan liposomes and F5-immunoliposomes of  Example 30 were used in this study. BT-474 subcutaneous  xenografts were raised in nude mice as in Example 29. At  day 18 after tumor cell inoculation the animals with tumors  (105-345 cubic mm, average about 200 cubic mm) were  randomized into treatment groups of 6 animals/group, and a  control group of 8 animals/group. The animals received free  or liposomal Topotecan at lxMTD, 0.5xMTD, 0.25xMTD,  or 0.125xMTD at three i.v. (tail vein) injections at day 19,  23, and 27 post tumor inoculation. The control group  received injections of physiological saline. The tumor sizes  and animal body weights were monitored as in Example 29.  Liposomal Topotecan was prepared generally following  the procedure of Example 18, using the entrapped solution  of TEA-SOS having 643 mM TEA, pH 5.7, osmolality 530  mmol/kg, and drug/phospholipid ratio of 170 mg/mmol. The  liposomes had 155 mg drug/mmol phospholipid; 90% load- ing efficiency, and particle size 105 nm. These liposomes  were incubated with the micellar solution ofF5-PEG-DSPE  conjugate at about 30 scFv per liposomes (15 mg antibody/  mmol phospholipid) at 60° C. for 1 hour generally as  described in Example 19. Antibody-conjugated liposomes  were separated by SEC using Sepharose CL-4B and formu› lated into HBS-6.5 HEPES-buffered saline. There was no  detectable change in drug/lipid ratio during the attachment  of anti-HER2 scFv (F5).  The uptake ofTopotecan formulations by cancer cells was  determined as follows. HER2-overexpressing human breast  adenocarcinoma cells (SK-Br-3, ATCC HTB-30) were  plated into 24-well cell culture plates at 150,000 cells/well  and acclimated overnight. The cells were incubated (in  triplicate) with PS-targeted and non-targeted liposomal  Topotecan in complete growth medium at liposome concen› trations of 0.1 mM and 0.01 mM for 4 hours at 37°  C. The  cells were rinsed 4 times with Hanks’ Balanced Salt Solu› tion, solubilized in 0.1% Triton X-100-70% acidified iso› propanol mixture  1:  10, and the amount of cell-associated  Topotecan per well was determined by fluorometry. The  results (mean–standard error) are summarized in Table 22.  The targeted liposomes delivered 100-300 times more drug  into the targeted cells than nontargeted liposomes.  TABLE 22  Maximum Tolerated Dose (MTD) of Free and  Liposomal Topotecan in Mice Given in 3 Weekly  i.v. Injections  Example 30  Example 32  Preparation and Targeted in vitro Cytotoxicity of  Topotecan Liposomes Prepared Using  Triethylammonium Sucrooctasulfate Entrapment  Method  Example 31  Antitumor Efficacy of Liposomal Topotecan in  BT-474 Xenograft Model at the Range of  0.125-1.0xMTD  Uptake of liposomal Topotecan by SK-Br-3 breast carcinoma cells.  Topotecan uptake at  Topotecan uptake at  0.1 mM phospholipid,  0.01 mM phospholipid,  Formulation  ng/well  ng/well  Non-targeted  4.76  – 0.24  0.607 – 0.088  liposome  HER2-targeted  533.8 – 13.7  197.0 – 4.6  liposome  Ratio: Targeted/  112.1  – 8.6  324 – 55  Non-targeted  To obtain animal body weight measurements, the tumor  weight ( calculated from the tumor size assuming tumor  density of 1.0) was subtracted from the total animal weight  measurements. All drug formulations at MTD showed anti- tumor activity (FIGS.  13A-13D).  There was no significant  difference in efficacy between free and liposomal drug given  at their respective MTD or at identical fractions (‰, …, or  $^{1}$/s) thereof. Thus, liposome encapsulation of the drug using  TEA-SOS gradient resulted in about 6-fold increase in  antitumor activity, but also in the similar increase in drug  toxicity. Dynamics of animal body weights revealed that all  treatments were non-toxic except the treatment with free  Topotecan at MTD which showed transient decrease in body  weight (about 15% of the pre-treatment value) that later  resolved (FIG.  14). 