US 9,717,723 B2  54  53  Example 23  in A-SOS liposomes was better than inA-DS liposomes both  in terms of magnitude (about 2-fold improvement in drug  retention) and statistical significance (statistical significance  at 95% confidence level by 2-tailed non-paired Student’s  t-test p=0.0257 and p=0.00995, respectively; and by Mann’s  U-test the difference was significant with a=0.01). Drug  retention in TEA-SOS containing Topotecan liposomes was  better than in A-SOS containing Topotecan liposomes.  The liposomes composed of DSPE, cholesterol, and PEG› DSPE (3:1:0.1 by weight), also containing [ $^{3}$H]-CHE at 0.22  mCi/mmol DSPE, were prepared as in Example 18, except  that the extrusion step included 10 passages through 2  stacked 200-nm pore filters, 10 passages through 2 stacked  100-nm pore filters, and 20 passages through 2 stacked  50-nm pore filters. The liposomes contained the following  salt solutions:  0.5 N ammonium dextran sulfate solution (A-DS) was  prepared from sodium dextran sulfate (M.w. 5000), pur› chased from Sigma, and converted into ammonium salt by  the ion exchange procedure similar to that of Example 4. The  solution of dextran sulfuric acid was immediately titrated  with 12.4 M aqueous ammonia. The A-DS solution has pH  5.66, osmolality 208 mmol/kg.  0.48 N ammonium sucrose octasulfate (A-SOS) was  prepared similar to Example 6, but ammonium hydroxide  was used for titration. The solution had pH 6.27, osmolality  258 mmol/kg.  0.47 M triethylammonium sucrose octasulfate (TEA› SOS) was prepared as in Example 6. The solution has pH  6.6, osmolality 297 mmol/kg.  Topotecan was loaded into the liposomes in the aqueous  solution of 10 mM MES-Na, 50 g/L dextrose, pH 6.5, by  incubating the liposomes with the drug at 61-62°  C. and  input drug/phospholipid ratio of 346–1 mg/mmol, for 40  min, followed by incubating on ice for 10 min. The lipo› somes were purified from unencapsulated drug by chroma› tography on Sephadex G-25, eluent-aqueous 2 mM Histi› dine, 144 mM NaCl, pH 6.6 (HCl).  Seven to nine week old female Swiss Webster mice were  injected via the tail vein with these liposomal Topotecan  formulations at the dose of 5 mg Topotecan per kg body  weight (approx. 0.2 mg Topotecan/animal) in triplicate.  After 8 hours or 24 hours post injection the blood was  collected and analyzed for Topotecan and liposome lipid as  in Example 22.  The results are presented in Table 17 below. While all  three liposome formulations demonstrated very close lipo› some circulation longevity, having about 23-28% of the  injected dose remaining in blood 24 hours post injection,  unexpectedly the drug retention in TEA-SOS liposomes and  The circulation longevity and Topotecan release param- eters were assessed in rats. The liposomes (DSPC/Choles› terol/PEG-DSPE molar ratio 3:2:0.015) were prepared by  ethanol mixing/extrusion method and loaded with Topotecan  using TEA-Pn gradient or TEA-Sucrose octasulfate (TEA› SOS) gradient as described in Example 18 and loaded at  various drug/lipid ratios (15-450 mg/mmol phospholipid).  For lipid matrix quantification, the liposome lipid contained  [3H]-CHE at 0.5-1.5 mCi/mmol DSPC. Female Sprague  Dawley rats (6-8 week old; body weight about 200 g) with  indwelling central venous catheters were injected i.v. (via  the catheter) with the Topotecan liposomes at the dose of 4-5  mg/kg of body weight. The catheter was flushed with saline.  At selected times (up to 48 hours post injection) the blood  samples (0.2-0.3 mL) were drawn via the catheter into  heparinized syringes, mixed with 0.4 mL of cold phosphate  buffered saline with 0.04% EDTA, blood cells were sepa- rated by centrifugation, and the supernatants (PBS-diluted  plasma) were assayed for lipid by  $^{3}$H-CHE radioactivity  counting ( quenching corrected), and for Topotecan by fluo› rometry (Example 71). The assay results were corrected for  plasma dilution, calculated from the weight of obtained  blood sample and assuming a hematocryt of 40%. The total  blood dose of the drug and lipid was estimated from the  blood volume calculated as 6.5% of the body weight. The  percent of Topotecan retained in the liposomes was calcu- lated by comparing the drug/lipid ratio at a given time point  to the drug/lipid ratio of the injected liposomes. Table 18  below summarizes blood half-lives of the lipid, the drug, and  the half-lives for drug release, as well as other properties of  the liposomes. Pharmacokinetic (PK) curves are shown on  FIGS.  SA  (lipid) and  SB  (drug/lipid ratio). In summary, the  blood PK curves for both drug and lipid fit well to single  exponent model (R 2  0.984-0.999). Despite their 90-100 nm  size and very small amount of PEGylated lipid (0.3 mo!.%),  the liposomes unexpectedly showed good circulation lon› gevity (plasma half-lives of the lipid component were in the  range of 11-16 hours). The slowest release of Topotecan  (half-time 22.9 hours) was observed with the liposomes  loaded using the TEA-SOS method.  Drug and Lipid Plasma Pharmacokinetics of  Liposomal Topotecan in Rats  Example 24  In vivo Drug Retention and Circulation Longevity  of Topotecan Liposomes Loaded Using Different  Entrapped Ammonium and Triethylammonium Salts  TABLE 17  In vivo drug retention and circulation persistence of Topotecan liposomes prepared  using TEA-SOS, ammonium-SOS (A-SOS), and ammonium dextran sulfate (A-DS.  Lipid remaining in  Topotecan remaining  circulation, %  encapsulated, %  Drug/phospho- Loading  injected dose  of initial load  lipid ratio,  efficiency,  Liposome  After 8  After 24  After 8  After 24  Gradient  mg/mmol  %  size, nm  hours  hours  hours  hours  A-DS  288.1  – 20.6  83.3 – 6.0  76.9 – 22.7  43.7 – 1.2 27.7 – 1.5 43.6 – 6.8 18.7 – 1.5  A-SOS  346.2  – 14.3  100.0 – 4.1  99.7 – 28.9  42.3 – 2.2 23.4 – 2.0 53.3 – 0.8 31.3 – 3.2  TEA-SOS  340.8  – 14.7  98.5 – 4.2  99.1 – 32.6 42.1 – 2.3 23.0 – 2.9 57.0 – 5.6 38.1 – 6.1 