METHODS FOR PREPARING LIPOSOMAL FORMULATIONS
Provided herein are methods of purifying liposomal formulations by washing with acidic aqueous solutions.
1 . A method of preparing a purified liposomal composition comprising:
liposomes comprising:
(a) a lipid bilayer;
(b) an internal medium; and
(c) a therapeutic agent encapsulated in the internal medium of the liposomes,
wherein the therapeutic agent has low water solubility and can be protonated to a protonated form;
said method comprising:
(i) providing a crude liposomal composition; and
(ii) purifying the crude liposomal composition with an acidified aqueous solution.
2 . The method of claim 1 , wherein:
the lipid bilayer comprises a first lipid and a first sterol;
the internal medium comprises a first loading aid.
3 . The method of claim 2 , wherein the first lipid is a polymer-conjugated lipid.
4 . The method of claim 2 or 3 , wherein the first lipid is selected from the group consisting of 1,2-distearoyl-rac-glycero-3-methoxypoly(ethylene glycol) (such as DSG-PEG2000), 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol) (such as DMG-PEG2000), 1,2-dipalmitoyl-rac-glycero-3-methoxypoly(ethylene glycol) (such as DPG-PEG2000), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (such as DSPE-PEG2000).
5 . The method of claim 4 , wherein the first lipid is DSG-PEG2000.
6 . The method of any one of claims 2 to 5 , wherein the lipid bilayer further comprises a second lipid.
7 . The method of claim 6 , wherein the second lipid is a phospholipid.
8 . The method of claim 6 or 7 , wherein the second lipid is distearoyl phosphatidyl choline (DSPC).
9 . The method of claim 6 or 7 , wherein the second lipid is hydrogenated sphingomyelin.
10 . The method of any one of claims 1 to 9 , wherein the internal medium is an aqueous internal medium.
11 . The method of claim 10 , wherein the aqueous internal medium is an acidic aqueous internal medium.
12 . The method of any one of claims 1 to 11 , wherein the internal medium further comprises an additional solvent.
13 . The method of claim 12 , wherein the additional solvent is an organic solvent.
14 . The method of claim 12 or 13 , wherein the additional solvent is dimethylsulfoxide (DMSO).
15 . The method of any one of claims 2 to 14 , wherein the first loading aid is an ionic loading aid.
16 . The method of any one of claims 2 to 15 , wherein the first loading aid is selected from the group consisting of ammonium sulfate (AS), ammonium sucrose octasulfate (NH4SOS), potassium sucrose octasulfate (KSOS), triethanolammonium sucrose octasulfate (TEA(OH)SOS), triethylammonium sucrose octasulfate (TEASOS), and sodium citrate.
17 . The method of any one of claims 2 to 16 , wherein the first loading aid is ammonium sulfate (AS).
18 . The method of any one of claims 2 to 16 , wherein the first loading aid is triethylammonium sucrose octasulfate (TEASOS).
19 . The method of any one of claims 2 to 18 , wherein the internal medium further comprises a second loading aid.
20 . The method of claim 19 , wherein the second loading aid is an ionic loading aid.
21 . The method of claim 19 or 20 , wherein the second loading aid is selected from the group consisting of ammonium sulfate (AS), ammonium sucrose octasulfate (NH4SOS), potassium sucrose octasulfate (KSOS), triethylammonium sucrose octasulfate (TEASOS), and sodium citrate.
22 . The method of any one of claims 19 to 21 , wherein the first loading aid is potassium sucrose octasulfate (KSOS) and the second loading aid is sodium citrate.
23 . The method of any one of claims 2 to 22 , wherein the first sterol is cholesterol or beta-sitosterol.
24 . The method of any one of claims 1 to 23 , wherein the therapeutic agent has a c Log P of greater than about 2.
25 . The method of any one of claims 1 to 24 , wherein the protonated form has a pKa of greater than about 2.
26 . The method of claim 24 or 25 , wherein the therapeutic agent is an anti-angiogenic agent, an antimetabolite, an apoptosis inducing agent, a cell cycle inhibitor, a cell cycle control inhibitor, a checkpoint inhibitor, a cyclin-dependent kinase inhibitor, a cytotoxic agent, a DNA damaging agent, a DNA repair inhibitor, a mitochondrial poison, a telomerase inhibitor, a signal transduction inhibitor, a transcription inhibitor, a Bcl inhibitor, a PARP inhibitor, a PI3K inhibitor, an HSP90 inhibitor, a JAK inhibitor, an ATR inhibitor, an HDAC inhibitor, a tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, a BTK inhibitor, an alkylating agent, an SMO inhibitor, an antitubulin agent, an MEK inhibitor, a topoisomerase inhibitor, a RAF inhibitor, a BRAF inhibitor, or a proteasome inhibitor.
27 . The method of claim 26 , wherein the therapeutic agent is a Bcl inhibitor selected from the group consisting of a Bcl-2 inhibitor, a Bcl-X L inhibitor, and a Bcl-2/Bcl-X L dual inhibitor.
28 . The method of any one of claims 1 to 27 , wherein the liposomes further comprise an additional therapeutic agent.
29 . The method of claim 28 , wherein the additional therapeutic agent is encapsulated in the liposomes.
30 . The method of claim 29 , wherein the additional therapeutic agent is an anti-angiogenic agent, an antimetabolite, an apoptosis inducing agent, a cell cycle inhibitor, a cell cycle control inhibitor, a checkpoint inhibitor, a cyclin-dependent kinase inhibitor, a cytotoxic agent, a DNA damaging agent, a DNA repair inhibitor, a mitochondrial poison, a telomerase inhibitor, a signal transduction inhibitor, a transcription inhibitor, a Bcl inhibitor, a PARP inhibitor, a PI3K inhibitor, an HSP90 inhibitor, a JAK inhibitor, an ATR inhibitor, an HDAC inhibitor, a tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, a BTK inhibitor, an alkylating agent, an SMO inhibitor, an antitubulin agent, an MEK inhibitor, a topoisomerase inhibitor, a RAF inhibitor, a BRAF inhibitor, or a proteasome inhibitor.
31 . The method of any one of claims 1 to 30 , wherein the liposomes have a mean diameter between about 50 nm and about 250 nm.
32 . The method of any one of claims 1 to 31 , wherein the acidified aqueous solution comprises a sugar.
33 . The method of any one of claims 1 to 32 , wherein the acidified aqueous solution comprises dextrose.
34 . The method of any one of claims 1 to 32 , wherein the acidified aqueous solution comprises sucrose.
35 . The method of any one of claims 1 to 32 , wherein the concentration of sugar in the acidified aqueous solution is between about 5 wt % and 20 wt %.
36 . The method of any one of claims 1 to 35 , wherein the concentration of acid in the acidified aqueous solution is between about 1 mM and 100 mM.
37 . The method of any one of claims 1 to 36 , wherein the acidified aqueous solution comprises methanesulfonic acid.