US 9,724,303 B2  52  51  The effect of drug to lipid ratio (0.15-0.45 mg/mmol  phospholipid) on the loading efficiency was also studied.  The liposomes with entrapped TEA-Pn (0.5 M TEA, pH 5.8,  osmolality 480 mmol/kg) were prepared as above, except  the final extrusion step was ten times through two stacked  0.08 µm  polycarbonate filters. The loading was at pH 6.5.  The liposome size was 93.1–15.1 nm by QELS. The results  (Table 14) showed that drug loading efficiency was over  85% over the whole range of drug/lipid ratios studied.  2.2-2.4 mL of Sepharose CL-2B, eluent HEPES-buffered  saline to separate the liposomes from plasma proteins and  free drug. The liposomes were collected in the void volume  fractions. Topotecan was quantified by fluorometry using  excitation at 384 nm and emission at 524 nm after solubi- lization of the plasma samples in 90% aqueous isopropanol- 0.1 N HCI, and the lipid was quantified by scintillation  counting of [ $^{3}$H]-CHE (quenching corrected). The deter› mined drug-to-lipid ratio at time was compared to the initial  ratio prior to incubation to obtain the % of Topotecan that  remained encapsulated at each time point. After 8 hours of  incubation, the amount of drug remaining in the liposome  was about 55% of its initial value (Table 15).  Liposomes (DSPC/Chol/PEG-DSPE, molar ratio 3:2:  0.015) with entrapped 0.5 N TEA-Pn, pH 6.2, osmolality  413 mmol/kg, were prepared as described in Example 18.  Liposomes with the size of96.4–29.3 nm were produced by  extrusion ten times through two stacked 100 nm pore size  polycarbonate filters. For quantitation of the liposome lipid  3  in plasma, [ HJ-CHE was included in the lipid solution at 0.5  µCi/µmol  ofDSPC. Topotecan was loaded at pH 6.0, 58°  C.  for 45 min at a drug/phospholipid ratio of 150 mg/mmol.  The efficiency of loading was 148.48–10.26 µg Topotecan/  µmo! phospholipid (99.0–6.8%).  The liposomes were incubated with 50% human plasma in  a multiwell microdialysis device (Spectra-Par MicroDia› lyzer 10-well, Spectrum, USA). Human donor plasma was  diluted by the equal volume of HEPES-buffered saline (20  mM HEPES, 135 mM NaCl), pH 6.5, containing 0.02%  sodium azide and charged into the lower reservoir of the  dialyzer (32 mL). The wells (0.4 mL) were separated from  the reservoir by a polycarbonate membrane with 30 nm pore  size, to afford free passage of plasma proteins and small  molecules but not the liposomes. The liposomes were mixed  with calculated amounts of plasma and HEPES-buffered  saline to achieve the concentration of 2.5 mM phospholipid  and 50 vol. % of plasma. The device was incubated at 37°  C., and the contents of the reservoir were stirred slowly.  After 8 hours of incubation, the contents of the lower  reservoir were changed for fresh 50% plasma. At the given  time points (see below) 50-µL  aliquots were withdrawn from  the wells, and chromatographed on the columns containing  The liposomes (DSPC/Chol/PEG-DSPE at 3:2:0.015  molar ratio, containing [ $^{3}$H]-CHE at 0.5 mCi/mmol DSPC)  with encapsulated gradient-forming salt solution (0.5 N  TEA-Pn, pH 6.2, osmolality 413 mmol/kg) were prepared as  in Example 18 using extrusion 12 times through two stacked  100 nm pore size polycarbonate filters. The liposome size  was 107.7–19.1 nm by QELS. The liposomes in 5 mM  HEPES, 50 g/L Dextrose, pH 6.5 were mixed with the  aqueous stock solution of Topotecan (20 mg/ml) at drug/  phospholipid ratios in the range 130-360 µg/µmol,  followed  my incubating the mixture at 58°  C. for 45 min, placing on  ice for 15 min and removal of unencapsulated drug by  Sephadex G-75 chromatography. Twelve-week old female  FvB mice were injected with the liposomes via the tail vein  at a dose of5 mg Topotecanperkg body weight (approx. 0.2  mg Topotecan/animal) in triplicate. At indicated times, typi- cally 8 hours or 24 hours post injection, the mice were  anesthetized, exsanguinated, and the blood samples were  assayed for the drug and the liposome lipid as in Example 8.  The results are shown in Table 16. After 24 hours, about  6-32% of the initial drug load remained encapsulated.  Higher loads of the drug (>200 mg/mmol phospholipid)  resulted in longer drug retention.  Example 21  Topotecan Liposome Stability In Vitro in the  Presence of Plasma  Example 22  Topotecan Liposomes with Entrapped TEA-Pn  Gradient at Various Drug/Lipid Ratios: In Vivo  Drug Retention and Circulation Longevity in Mice  TABLE 14  TABLE 15  Effect of drug/lipid ratio on the encapsulation efficiency  ofTopotecan into the liposomes containing TEA-Pn.  Topotecan/phospholipid ratio, mg/mmol  Input ratio  Output ratio ( after loading)  168.2  166.9 – 11.1  224.4  232.5 – 47.6  280.3  253.5 – 19.8  336.4  298.3 – 18.0  392.5  361.2 – 36.8  448.5  394.9 – 29.5  % encapsulation  (mean– SE)  99.2  – 6.6  103.7  – 21.2  90.4 7.0  88.7  – 5.3  92.0 – 9.4  88.0 – 6.6  In vitro release of Topotecan from the liposomes loaded  by TEA-Pn gradient in 50% human plasma at 37°  C.  Incubation time, hours  4  8  24  % drug remaining encapsulated  95.5 – 5.4  76.8  – 7.3  55.9  – 4.1  55.4  – 16.8 