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## In vivo Drug Retention and Circulation Longevity of Topotecan Liposomes Loaded Using Different Entrapped Ammonium and Triethylammonium Salts 

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 

## Example 23 

<img src='content_image/938125.jpg'>

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. 

## Drug and Lipid Plasma Pharmacokinetics of Liposomal Topotecan in Rats 

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. 

## Example 24 

