Thermosensitive Nanoparticle Formulations and Method of Making The Same

Abstract

The present invention relates to a formulation of thermosensitive liposomes, and more specifically to a formulation of liposomes comprising phospholipids and a surface active agent, wherein the liposomes support long term storage at temperatures less than or equal to about 8° C., control degradate formation to maximize product potency and release their contents at mild hyperthermic temperatures. Methods of making formulations are also described.

What is claimed is: 
     
         1 . A liposomal preparation, comprising a suspension of liposomes having a gel-phase lipid bilayer and doxorubicin entrapped inside the liposomes; said lipid bilayer comprising:
 (i) one or more phospholipids selected from the group consisting of phosphatidyl cholines, phosphatidyl glycerols, phosphatidyl inositols, and phosphatidyl ethanolamines;   (ii) one or more phospholipids derivatized with a hydrophilic polymer; and   (iii) one or more lysolipids selected from the group consisting of monoacylphosphatidyl cholines, monoacylphosphatidylglycerols, monoacylphosphatidylinositols, and monoacylphosphatidyl-ethanolamines;   wherein the lipid bilayer constituents are provided in a molar ratio of about 80-90:2-8:2-18; and wherein the size of the liposomes in the suspension is between about 50 and about 150 nm; and   wherein the relative concentration of impurity A after 6 months of storage at less than or equal to 8° C. is less than 0.5%, and wherein impurity A is a peak with a relative retention time approximately 1.4 in a high performance liquid chromatography (HPLC) with a C18 reverse phase column with an acetic acid/methanol solvent gradient elution conditions.   
     
     
         2 . The liposomal preparation of  claim 1 , wherein the relative concentration of impurity A after about 1 year of storage at less than or equal to 8° C. is less than about 0.5%. 
     
     
         3 . The liposomal preparation of  claim 1 , wherein the relative concentration of impurity A after about 2 years of storage at less than or equal to 8° C. is less than about 0.75%. 
     
     
         4 . The liposomal preparation of  claim 1 , wherein the relative concentration of 8-desacetyl-8-carboxy daunorubicin after about 1 year of storage at less than or equal to 8° C. is less than about 0.5%. 
     
     
         5 . The liposomal preparation of  claim 1 , wherein the relative concentration of 8-desacetyl-8-carboxy daunorubicin after about 2 years of storage at less than or equal to 8° C. is less than about 1.6%. 
     
     
         6 . The liposomal preparation  claim 1 , wherein the concentration of doxorubicin after about one year of storage at a temperature of about less than or equal to 8° C. is greater than 97% of the initial doxorubicin concentration, as determined by HPLC with a C18 reverse phase column with an acetic acid/methanol solvent gradient elution conditions. 
     
     
         7 . The liposomal preparation of  claim 1 , wherein the concentration of doxorubicin after about two years of storage at a temperature of about less than, or equal to 8° C. is greater than 95% of the initial doxorubicin concentration, as determined by HPLC with a C18 reverse phase column with an acetic acid/methanol solvent gradient elution conditions. 
     
     
         8 . The liposomal preparation of  claim 1 , wherein the formation of total degradation products after about one year of storage at a temperature of about less than or equal to 8° C. is less than 1%. 
     
     
         9 . The liposomal preparation of  claim 1 , wherein the formation of total degradation products after about two years of storage at a temperature of about less than or equal to 8° C. is less than 2.5%. 
     
     
         10 . The liposomal preparation of  claim 1 , wherein the one or more phospholipids is dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidyl glycerol (DSPC), or a combination thereof; the lysolipid is monopalmitoylphosphaticlylcholine (MPPC), monolaurylphosphatidylcholine (MLPC), monomyristoylphosphatidylcholine (MMPC), monostearoylphosphatidylcholine (MSPC), or mixtures hereof; and the one or more phospholipids derivatized with a hydrophilic polymer is a PEGylated lipid. 
     
     
         11 . The liposomal preparation of  claim 1 , wherein the one or more phospholipids is dipalmitoylphosphatidylcholine, one or more phospholipids derivatized with a hydrophilic polymer is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol) 2000], and the one or more lysolipids is monostearoylphosphatidylcholine. 
     
     
         12 . The liposomal preparation of  claim 1 , further comprising an imaging agent or a diagnostic agent entrapped in the liposome. 
     
     
         13 . The liposomal preparation of  claim 12 , wherein the imaging agent or diagnostic agent is an X-ray contrast agent, an MRI contrast agent, an ultrasonic imaging agent, a fluorescent agent or a radioactive agent. 
     
     
         14 . A method for loading doxorubicin into temperature sensitive liposomes, comprising:
 (a) preparing a suspension of liposomes having a gel-phase lipid bilayer and a greater concentration of ammonium ions inside the liposomes than outside the liposomes, said lipid bilayer comprising:
 (i) one or more phospholipids selected from the group consisting of phosphatidyl cholines, phosphatidyl glycerols, phosphatidyl inositols, and phosphatidyl ethanolamines; 
 (ii) one or more phospholipids derivatized with a hydrophilic polymer; and 
 (iii) one or more lysolipids selected from the group consisting of monoacylphosphatidyl cholines, monoacylphosphatidylglycerols, monoacylphosphatidylinositols, and monoacylphosphatidyl-ethanolamines; 
   wherein the lipid bilayer constituents are provided in a molar ratio of about 80-90:2-8:2-18; and   where said preparing includes reducing the size of the liposomes in the suspension to an average particle size of between about 50 and about 150 nm;   (b) adding a doxorubicin solution to the suspension of liposomes, wherein the doxorubicin is taken up into the liposomes.   
     
     
         15 . The method of  claim 14 , wherein at least 95% of the doxorubicin present in the solution is taken up into the liposomes. 
     
     
         16 . The method of  claim 14 , wherein the concentration of doxorubicin taken up into the liposomes is about 50 mM to about 75 mM. 
     
     
         17 . The method of  claim 14 , wherein said preparing comprises preparing the liposomes in the presence of an ammonium sulfate solution. 
     
     
         18 . The method of  claim 17 , wherein the concentration of ammonium sulfate is about 100 mM to about 300 mM. 
     
     
         19 . The method of  claim 18 , further comprising replacing the ammonium ions outside the liposomes with a monosaccharide or disaccharide solution. 
     
     
         20 . The method of  claim 19 , wherein the concentration of the monosaccharide or disaccharide solution is about 5-15%. 
     
     
         21 . The method of  claim 20 , wherein the ammonium ions outside the liposomes are replaced with a monosaccharide solution. 
     
     
         22 . The method of  claim 21 , wherein the monosaccharide solution is a lactose solution. 
     
     
         23 . The method of  claim 14 , further comprising adding a histidine buffer after step b). 
     
     
         24 . The method of  claim 23 , wherein the concentration of the histidine buffer is about 5 mM to about 15 mM. 
     
     
         25 . The method of  claim 14 , wherein the one or more phospholipids have two same or different C 14 -C 20  acyl groups. 
     
     
         26 . The method of  claim 25 , wherein the one or more phospholipids is dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidyl glycerol (DSPG), or a combination thereof. 
     
     
         27 . The method of  claim 14 , wherein the lysolipid is monopalmitoylphosphatidylcholine (MPPC), monolaurylphosphatidylcholine (MLPC), monomyristoylphosphatidylcholine (MMPC), monostearoylphosphatidylcholine (MSPC), or mixtures thereof. 
     
     
         28 . The method of  claim 14 , wherein the one or more phospholipids derivatized with a hydrophilic polymer is a PEGylated lipid. 
     
     
         29 . The method of  claim 14 , wherein the one or more phospholipids is dipalmitoylphosphatidylcholine, one or more phospholipids derivatized with a hydrophilic polymer is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol) 2000], and the one or more lysolipids is monostearoylphosphatidylcholine. 
     
     
         30 . A liposome preparation made by the method of  claim 14 .