Vitro release assay for liposome encapsulated vincristine

Abstract

The present invention provides a novel in vitro release assay for the accurate, reproducible quantitation of vincristine sulfate release from liposomes.

1 . A commercially marketable liposomal vincristine sulfate solution prepared by a method comprising the steps of:
 a. adding VSLI to an incubation solution comprising 1-butanol and phosphate buffered saline (PBS), wherein the concentration of 1-butanol after addition of the VSLI is between about 2.7% and about 2.8% (v/v);   b. heating the incubation solution containing the VSLI for at least about 72 hours at about 37° C.±0.1° C.;   c. removing an aliquot of the incubation solution containing the VSLI at two or more time points;   d. separating the released vincristine from the VSLI by filtering each aliquot through a membrane which retains particles with D25 of 70 nm and D90 of 170 nm separate; and   e. determining the amount of released vincristine in the filtrate of each aliquot removed at each time point by high performance liquid chromatography,
 wherein the difference in the amount of released vincristine at each time point is indicative of the release rate of vincristine from the VSLI. 
   
     
     
         2 . The liposomal vincristine sulfate solution of  claim 1 , wherein the concentration of 1-butanol in the incubation solution is 2.75%. 
     
     
         3 . The liposomal vincristine sulfate solution of  claim 1 , wherein the VSLI is diluted by about 1:21 in the incubation solution. 
     
     
         4 . The liposomal vincristine sulfate solution of  claim 1 , wherein the pH of the incubation solution is between about 7.0 and about 7.4. 
     
     
         5 . The liposomal vincristine sulfate solution of  claim 1 , wherein the pH of the solution is about 7.4. 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
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         11 . The liposomal vincristine sulfate solution of  claim 1 , wherein the nominal molecular weight limit of the membrane is between about 10,000 and 100,000 daltons. 
     
     
         12 . The liposomal vincristine sulfate solution of  claim 1 , wherein the nominal molecular weight limit of the membrane is between about 30,000 and 100,000 daltons. 
     
     
         13 . The liposomal vincristine sulfate solution of  claim 1 , wherein the nominal molecular weight limit of the membrane is about 100,000 daltons. 
     
     
         14 . (canceled) 
     
     
         15 . The liposomal vincristine sulfate solution of  claim 1 , wherein the membrane is pretreated with a passivation solution comprising bovine serum albumin, polyethylene glycol and PBS. 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . The liposomal vincristine sulfate solution of  claim 1 , wherein greater than about 80% of the vincristine encapsulated in the VSLI is released within 72 hours. 
     
     
         26 . (canceled) 
     
     
         27 . The liposomal vincristine sulfate solution of  claim 1 , wherein the VSLI comprises a concentration ratio of about 0.1/1.0 to about 0.2/2.0 vincristine sulfate to lipid. 
     
     
         28 . The liposomal vincristine sulfate solution of  claim 1 , wherein the concentration of vincristine sulfate in the VSLI is about 0.1 mg/mL to about 0.5 mg/mL. 
     
     
         29 . The liposomal vincristine sulfate solution of  claim 1 , wherein the concentration of vincristine sulfate in the VSLI is about at about 0.15 mg/mL to about 0.2 mg/mL. 
     
     
         30 . The liposomal vincristine sulfate solution of  claim 1 , wherein the concentration of vincristine sulfate in the VSLI is about at about 0.16 mg/mL. 
     
     
         31 . The liposomal vincristine sulfate solution of  claim 1 , wherein the liposomes in the VSLI comprise between about 75/25 mol %/mol sphingomyelin/cholesterol to 30/50 mol %/mol % sphingomyelin/cholesterol. 
     
     
         32 . The liposomal vincristine sulfate solution of  claim 1 , wherein the liposomes in the VSLI comprise about 70/30 mol %/mol sphingomyelin/cholesterol to 40/45 mol %/mol % sphingomyelin/cholesterol. 
     
     
         33 . The liposomal vincristine sulfate solution of  claim 1 , wherein the liposomes in the VSLI comprise approximately 55/45 mol %/mol % sphingomyelin/cholesterol. 
     
     
         34 . The liposomal vincristine sulfate solution of  claim 1 , wherein the liposomes have a mean diameter of about 90-125 nm. 
     
     
         35 . A vincristine sulfate liposomal injection solution assayed according to the method of  claim 1 . 
     
     
         36 . A batch of vincristine sulfate liposomal injection solution assayed according to the method of  claim 1 . 
     
     
         37 . (canceled)