Liposomal formulation of nonglycosidic ceramides and uses thereof

Abstract

The invention provides liposomes containing nonglycosidic ceramides within their bilayers, and compositions thereof. These liposomes activate murine iNKT cells and induce dendritic cell (DC) maturation, both in vitro and in vivo at an efficacy that is comparable to their corresponding soluble nonglycosidic ceramides. Also provided are methods for treating diseases using the liposomes and compositions of the invention.

1 .- 114 . (canceled) 
     
     
         115 . A liposome comprising:
 (a) threitolceramide present in an amount of about 3 wt. % to about 12 wt. %, based upon the total weight of the liposome;   (b) a first lipid present in an amount of about 20 wt. % to about 30 wt. %, based upon the total weight of the liposome; and   (c) a second lipid present in an amount of about 65 wt. % to about 75 wt. %, based upon the total weight of the liposome.   
     
     
         116 . The liposome of  claim 115 , wherein the first lipid comprises a phosphotidylcholine. 
     
     
         117 . The liposome of  claim 116 , wherein the phosphatidylcholine is selected from the group consisting of 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), egg phosphatidylcholine (EPC), and mixtures thereof. 
     
     
         118 . The liposome of  claim 117 , wherein the phosphtidylcholine comprises egg phosphatidylcholine (EPC). 
     
     
         119 . The liposome of  claim 115 , wherein the second lipid comprises phosphatidylglycerol. 
     
     
         120 . The liposome of  claim 119 , wherein the phosphatidylglycerol is selected from the group consisting of 1,2-dierucoyl phosphatidylglycerol (DEPG), 1,2-dilauroyl phosphatidylglycerol (DLPG), 1,2-dimyristoyl phosphatidylglycerol (DMPG), 1,2-dioleoyl phosphatidylglycerol (DOPG), 1,2-dipalmitoyl phosphatidylglycerol (DPPS), 1,2-distearoyl phosphatidylglycerol (DSPG), 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), egg phosphatidylglycerol (EPG), salts of any of the foregoing, and mixtures thereof. 
     
     
         121 . The liposome of  claim 120 , wherein the phosphatidylglycerol comprises egg phosphatidylglycerol (EPG). 
     
     
         122 . The liposome of  claim 115 , further comprising at least one antigen. 
     
     
         123 . The liposome of  claim 122 , wherein the at least one antigen comprises at least one tumor antigen. 
     
     
         124 . The liposome of  claim 123 , wherein the tumor antigen comprises a member selected from the group consisting of P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, XAGE, antigenic fragments thereof, and mixtures thereof. 
     
     
         125 . The liposome of  claim 115 , further comprising at least one adjuvant. 
     
     
         126 . The liposome of  claim 115 , further comprising at least one therapeutic agent or antigen. 
     
     
         127 . The liposome of  claim 126 , wherein the at least one therapeutic agent or antigen is selected from the group consisting of an immune modulator, a Toll-like receptor agonist, a Nod ligand, an anti-viral agent, an antifungal agent, an antibiotic, an antiviral antibody, a cancer immune therapeutic, a chemotherapy agent, a kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an antihistamine agent, an anti-inflammatory agent, a vaccine adjuvant, a second liposome, an artificial antigen presenting cell, a cytokine or chemokine blocking antibody, P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, XAGE, antigenic fragments thereof, and combinations thereof. 
     
     
         128 . The liposome of  claim 115 , having a diameter of less than about 100 nm. 
     
     
         129 . A composition comprising the liposome of  claim 115  and a pharmaceutically acceptable diluent, excipient or carrier. 
     
     
         130 . The composition of  claim 129 , wherein the liposome is present in the composition in an amount of about 1 mg/mL to about 20 mg/mL. 
     
     
         131 . The composition of  claim 129 , wherein the composition is formulated for parenteral, intrathecal, transdermal, rectal, oral, or nasal administration. 
     
     
         132 . The composition of  claim 129 , further comprising at least one therapeutic agent. 
     
     
         133 . The composition of  claim 132 , wherein the therapeutic agent is within the liposome. 
     
     
         134 . A method of stimulating an immune response in a mammalian subject comprising administering to the subject the liposome of  claim 115 . 
     
     
         135 . The method of  claim 134 , further comprising administering a therapeutic agent to the mammalian subject. 
     
     
         136 . A method of treating a mammalian subject having cancer comprising administering to said subject a therapeutically effective amount of the liposome of  claim 115 . 
     
     
         137 . The method of  claim 136 , wherein the cancer is selected from the group consisting of basal cell carcinoma, breast cancer leukemia, Burkitt's lymphoma, colon cancer, esophageal cancer, bladder cancer, gastric cancer, head and neck cancer, hepatocellular cancer, Hodgkin's lymphoma, hairy cell leukemia, Wilms' tumor, thyroid cancer, thymoma and thymic carcinoma, testicular cancer, T-cell lymphoma, prostate cancer, non-small cell lung cancer, liver cancer, renal cell cancer, melanoma, and combinations thereof. 
     
     
         138 . The method of  claim 136 , further comprising administering to the subject at least one further therapeutic agent, therapy, or antigen selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent, radiation therapy, an immune modulator, a Toll-like receptor agonist, a Nod ligand, an anti-viral agent, an antifungal agent, an antibiotic, an antiviral antibody, a cancer immune therapeutic, a chemotherapy agent, a kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an antihistamine agent, an anti-inflammatory agent, a vaccine adjuvant, a second liposome, an artificial antigen presenting cell, a cytokine or chemokine blocking antibody, P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-All, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, XAGE, antigenic fragments thereof, and a combination thereof. 
     
     
         139 . A method of reducing the growth or metastatic spread of a tumor in a mammalian subject comprising administering to said subject a therapeutically effective amount of a combination therapy comprising the liposome of  claim 115  or a composition comprising the liposome and at least one further therapeutic agent, therapy, or antigen selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent, radiation therapy, an immune modulator, a Toll-like receptor agonist, a Nod ligand, an anti-viral agent, an antifungal agent, an antibiotic, an antiviral antibody, a cancer immune therapeutic, a chemotherapy agent, a kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an antihistamine agent, an anti-inflammatory agent, a vaccine adjuvant, a second liposome, an artificial antigen presenting cell, a cytokine or chemokine blocking antibody, P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-All, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, XAGE, antigenic fragments thereof, and a combination thereof. 
     
     
         140 . A method of treating an infection in a subject, said infection caused by an infectious agent selected from the group consisting of a virus, a microbe or bacteria, or a parasite, comprising administering to said subject a therapeutically effective amount of the liposome of  claim 115 . 
     
     
         141 . A method of making the liposome of  claim 115  comprising:
 (a) preparing a stock solution of threitolceramide in an organic solvent; 
 (b) combining an aliquot of the stock solution with a mixture containing a first lipid and a second lipid to form a lipid solution; 
 (c) diluting the lipid solution with an aqueous solution; 
 (d) forming multi-lamellar vesicles (MLVs) from the diluted lipid solution; and 
 (e) downsizing the MLVs to about 50 nm to about 150 nm at a temperature above the Tc of the lipids, 
 
       wherein the threitolceramide is present in an amount of about 3 wt. % to about 12 wt. %, based upon the total weight of the liposome; the first lipid is present in an amount of about 20 wt. % to about 30 wt. %, based upon the total weight of the liposome; and the second lipid present in an amount of about 65 wt. % to about 75 wt. %, based upon the total weight of the liposome.