Method of lyophilizing liposomes

Abstract

Lyophilized liposomal formulations with two or more encapsulated drugs are disclosed. These formulations display superior drug retention profiles and also maintain size distribution following lyophilization and reconstitution.

1 . A method to administer therapeutic and/or diagnostic agents to an animal subject which method comprises administering to said subject a reconstituted formulation of a lyophilized composition, wherein the lyophilized composition has been reconstituted in a pharmaceutical carrier, which lyophilized composition comprises:
 (a) gel-phase liposomes that exhibit a melting phase temperature (T c ) of at least 37° C., wherein the liposome membrane of said liposomes comprises no more than 20 mol % cholesterol and at least 1 mol % of a phosphatidylglycerol (PG) or a phosphatidylinositol (PI) or both, wherein at least two therapeutic and/or diagnostic agents are stably associated with said liposomes, wherein at least one of said agents is amphipathic or hydrophilic; and;   (b) a cryoprotectant external to said liposomes; and   
       wherein said liposomes contain substantially no internal cryoprotectant, and
 wherein when said lyophilized gel-phase liposomal composition is reconstituted in said pharmaceutical carrier, the mean diameter of the liposomes is maintained as compared to said composition prior to lyophilization and said agents are substantially retained in the liposomes. 
 
     
     
         2 . The method of  claim 1  wherein said stably associated agents are at a fixed ratio and wherein when said composition is reconstituted said ratio of the agents changes by no more than 25% as compared to said composition prior to lyophilization. 
     
     
         3 . The method of  claim 1  wherein the stably associated agents are antineoplastic agents. 
     
     
         4 . The method of  claim 3  wherein the antineoplastic agents are daunorubicin and cytarabine, or are irinotecan and floxuridine. 
     
     
         5 . The method of  claim 4  wherein the stably associated agents are daunorubicin:cytarabine at a fixed ratio of 1:5. 
     
     
         6 . The method of  claim 1  wherein the liposome membrane comprises 50-80 mol % DSPC. 
     
     
         7 . The method of  claim 1  wherein the mean diameter of the liposomes increases by no more than 25% after lyophilization and upon reconstitution of said liposomes as compared to said value measured prior to lyophilization. 
     
     
         8 . The method of  claim 1  wherein said mean diameter of the liposomes is maintained for at least 6 months upon storage at from 5° C. to 25° C. 
     
     
         9 . The method of  claim 1  wherein at least 75% of each stably associated agent is retained upon reconstitution of said liposomes. 
     
     
         10 . The method of  claim 1  wherein the size distribution of the liposomes changes by no more than 25% after lyophilization and upon reconstitution of said liposomes. 
     
     
         11 . The method of  claim 1  wherein said administering is parenteral. 
     
     
         12 . The method of  claim 1  wherein the subject is human. 
     
     
         13 . The method of  claim 3  wherein the subject is diagnosed with cancer. 
     
     
         14 . The method of  claim 4  wherein the subject is diagnosed with cancer. 
     
     
         15 . The method of  claim 5  wherein the subject is diagnosed with cancer. 
     
     
         16 . The method of  claim 1 , wherein the liposome membrane comprises di stearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), and cholesterol (CHOL). 
     
     
         17 . The method of  claim 1 , wherein the liposome membrane comprises 50-80 mol % DSPC or dipalmitoyl phosphatidylcholine (DPPC), 1-20 mol % DSPG or distearoylphosphatidylinositol (DSPI) and 1-20 mol % CHOL. 
     
     
         18 . The method of  claim 17 , wherein the liposome membrane comprises 50-80 mol % DSPC, 1-20 mol % DSPG and 1-20 mol % CHOL. 
     
     
         19 . The method of  claim 18 , wherein the liposome membrane has the components DSPC:DSPG:CHOL at 7:2:1 mol ratio.