US 8,992,970 B2  76  75  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.  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.  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).  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  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%  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.  Example 56  Preparation and Pharmacokinetics of Doxorubicin  Encapsulated in the Liposomes Using  Triethylammonium Sulfate Method  In Vivo Antitumor Efficacy of EGFR-Targeted  Liposomal Vinorelbine Against U87 Human Brain  Cancer Xenograft Tumors in Mice  Example 57  Doxorubicin-Loaded Liposomes andAnti-HER2  Immunoliposomes Prepared by TEA-Sulfate  Method: Preparation and In Vivo Antitumor Efficacy  Against HER2-Overexpressing Human Breast  Cancer Xenografts  TABLE30  Properties of liposomal doxorubicin at various  lipid  compositions.  Lipid composition (molar ratio)  DSPC/Chol/PEG-DSPE (3:2:0.015)  DSPC/Chol (3:2)  Liposome size, nm  (mean– SD  by QELS)  drug/  phospholipid  (mg/mmol)  DSPC/Chol/Glu-DSPE (2.85:2:0.15)  DSPC/Chol/PEG-DSPE (2.7:2:0.3)  81.8 – 27.3  79.1 – 27.9  83.6 – 27.2  83.7 –23.1  163.6 – 4.4  137.0 – 17.5  141.7 – 10.4  175.0 – 6.8  Example 55 