content_image/947751.jpg
into the liposomes at the drug/phospholipid ratio of 350 mg/mmol. F5 immunoliposomal vinorelbine was formed by incubating these liposomes with F5-PEG-DSPE conjugate (Example 19) as described in Example 43. Nude mice with BT-4 7 4 xenografts were raised and treated i.v. with free VRB, 5 F5-ILs-VRB-"0.5% PEG", or F5-ILs-VRB-"10% PEG" at 5 mg/kg as in Example 53. As shown in FIG. 34, F5-ILs-VRB with higher PEGylation provided with a non-ionic PEG lipid derivative PEG-DSG was noticeably more efficacious in reducing tumor growth than F5-ILs-VRB with lower amount 10 of PEG-DSG, while both preparations were more active than the free drug. 

## In Vivo Antitumor Efficacy of EGFR-Targeted Liposomal Vinorelbine Against U87 Human Brain Cancer Xenograft Tumors in Mice 

The liposomes (86.6–12.9 nm in size by QELS) with encapsulated 0.65 M TEA-SOS solution were prepared and loaded with VRB according to Example 42. Anti-EGFR› immunoliposomal VRB (C225Fab’-ILs-VRB) was prepared by incubation ofVRB liposomes with the PEG-DSPE conju› gate ofan anti-EGFR antibody Fab’ fragments as described in Example 36. 

Male NCR nu/nu mice (5-6 weeks, weighing 17-20 g) were injected subcutaneously in the flank area with lxl0 $^{7 }$ofU87 human glioblastoma cells (ATCC) suspended in the growth medium in a total volume of 150 µI. When the tumor reached an average size of250 mm 3 , mice were randomly divided into four groups of 10-12 animals. The mice were treated with three weekly i.v. injections of "free" VRB, nontargeted Ls› VRB, or C225Fab’-ILs-VRB at a dose of 5 mg VRB/kg. The control group received an equal volume of saline. The tumor sizes and animal body weights were monitored as in Example 10. C225-Fab’-ILs-VRB was noticeably more efficacious in suppressing the growth of EGFR-overexpressing human brain cancer xenograft tumors than either non-targeted lipo› somal vinorelbine or free vinorelbine at an equal dose (FIG. 35). 

## Preparation and Pharmacokinetics of Doxorubicin Encapsulated in the Liposomes Using Triethylammonium Sulfate Method 

Liposomes with various lipid matrix composition ( as indi› cated in the table below) were formed as described in Example 2. N-Glutaryl-DSPE (Glu-DSPE) was fromAvanti Polar Lipids, AL, USA. A neat lipid film was formed from the lipid solution in chloroform using rotary evaporation, trace volatiles were removed under vacuum (90 µm Hg, 2 hours), the lipid film was hydrated in a triethylanimonium sulfate (TEA-SO$_{4}$ ) solution (0.65 N TEA), subjected to six cycles of rapid freeze and thaw, and extruded through two stacked O .1 µm pore size polycarbonate filters ten times and through two stacked 0.05 µm pore size polycarbonate filters ten times. For lipid matrix quantification in the blood samples, [ $^{3}$H]-CHE was included in the lipid matrix at 0.5-1.5 mCi/mmol phos› pholipid. The liposomes with entrapped TEA-SO$_{4 }$ solution were loaded with doxorubicin according to Example 2. The liposomes in HEPES-buffered saline (20 mM HEPES-Na, 135 mM NaCl, pH 6.5) were incubated with doxorubicin hydrochloride ( drug/phospholipid ratios of 140-170 mg/mmol) at 60° C. for 45 min followed by quenching on ice 

## Example 55 

## Example 56 

and removal ofunencapsulated doxorubicin by gel chroma› tography. Doxorubicin was assayed by spectrophotometry (Example 71), and phospholipid was assayed by Bartlett method (Example 70). The properties of resulting liposomes are sUlllillarized in Table 30 below. 

<img src='content_image/947751.jpg'>

Blood pharmacokinetics of these doxorubicin-containing liposomes having lipid composition of DSPC/Chol/PEG› DSPE 2.7:2:0.3 was studied in rats at a single i.v. dose of5 mg doxorubicin/kg as described in Example 9. The liposomes were long circulating (half-life of about 28 hours) (FIG. 36). The stable doxorubicin-to-phospholipid ratio indicated that the formulation was remarkably stable against the drug leak› age in the circulation, losing less than 25% of the drug over a 48-hour time period. 

## Doxorubicin-Loaded Liposomes andAnti-HER2 Immunoliposomes Prepared by TEA-Sulfate 

## Method: Preparation and In Vivo Antitumor Efficacy Against HER2-Overexpressing Human Breast Cancer Xenografts 

Doxorubicin-loaded liposomes having various lipid com- positions and properties (listed in the table below) were pre› pared as described in Example 56. Doxorubicin-loaded anti› HER2 immunoliposomes were prepared from doxorubicin› loaded liposomes by co-incubation with anti-HER2 scFv F5-PEG-DSPE conjugate (approx. 30 scFv/liposome) as described in Example 19. NCR nu/nu mice bearing the sub› cutaneous human breast tumor xenograft (BT-474) were raised, treated (in groups of 10-12 animals) with liposomal or anti-HER2 immunoliposomal doxorubicin at a dose of 5 mg/kg once weekly for a total of three weeks once the tumors reached an average size of 200 mm 3 , and the tumor progres› sion and animal body weights were monitored as described in Example 29. For non-targeted doxorubicin liposome formu› lations, the lipid compositions containing no PEG-DSPE, 0.5 mo!.% PEG-DSPE, or 10 mo!.% PEG-DSPE, were studied; for F5-immunoliposomal doxorubicin, the formulations with 0.5 mo!. % PEG-DSPE and 10 mo!. % PEG-DSPE were studied (here the quantity of PEG-DSPE is expressed as mo!. % ofliposome phospholipid). The results (FIG. 37, Table 31) demonstrated that all doxorubicin treatments were effective in retarding the tumor growth. On the basis of tumor sizes at day 53 post inoculation, the differences in tumor growth inhibition among all three non-targeted liposome groups did not raise to statistical significance (AN OVA p=0.081 ), but the immunoliposome doxorubicin was significantly more effica› cious than non-targeted liposomal doxorubicin (ANOVA p=5. 5 x 10- 10 , the "10% PEG-DSPE" formulation being more efficacious than "0.5% PEG-DSPE" (Student’s t-test, p=0.027). In the") 0% PEG-DSPE" F5-ILs group, the tumors regressed to 1 mm $^{3 }$or less in 67% of animals, while in "0.5% 

## Example 57 

