Amphoteric Liposome Formulation

Abstract

The invention relates to compositions and methods to inhibit gene expression. In particular, the invention provides DNAi oligonucleotides sequestered by amphoteric liposomes for the treatment of cancer.

What is claimed is: 
     
         1 . A mixture comprising an amphoteric liposome and a DNAi oligonucleotide. 
     
     
         2 . The mixture of  claim 1 , wherein the amphoteric liposome has an isoelectric point between 4 and 8. 
     
     
         3 . The mixture of  claim 1  or  2 , wherein the amphoteric liposome has a net negative charge or are neutral at pH 7.4 and have a net positive charge at pH 4. 
     
     
         4 . The mixture of  claim 1 ,  2  or  3 , wherein the amphoteric liposome a is formed from a lipid phase comprising an amphoteric lipid. 
     
     
         5 . The mixture of  claim 4 , wherein the lipid phase comprises 5 to 30 mol. % of the amphoteric lipid. 
     
     
         6 . The mixture of  claim 4  or  5 , wherein the amphoteric lipid is selected from the group consisting of HistChol, HistDG, isoHistSuccDG, Acylcarnosine and HCChol. 
     
     
         7 . The mixture of  claim 1 ,  2  or  3 , wherein the amphoteric liposome is formed from a lipid phase comprising a mixture of lipid components with amphoteric properties. 
     
     
         8 . The mixture of  claim 7 , wherein the mixture of lipid components comprises anionic or cationic components, wherein at least one such component is pH responsive. 
     
     
         9 . The mixture of  claim 8 , wherein the mixture of lipid components are selected from the group consisting of (i) a stable cationic lipid and a chargeable anionic lipid, (ii) a chargeable cationic lipid and chargeable anionic lipid and (iii) a stable anionic lipid and a chargeable cationic lipid. 
     
     
         10 . The mixture of  claim 9 , wherein the lipid components comprise one or more anionic lipids selected from the group consisting of DOGSucc, POGSucc, DMGSucc, DPGSucc, DGSucc, DMPS, DPPS, DOPS, POPS, DMPG, DPPG, DOPG, POPG, DMPA, DPPA, DOPA, POPA, CHEMS and Cet-P. 
     
     
         11 . The mixture of  claim 9  or  claim 10 , wherein the lipid components comprise one or more anionic lipids selected from the group consisting of DMGSucc, DOPA, CHEMS and Cet-P. 
     
     
         12 . The mixture of any of  claims 8  to  11 , wherein the lipid components comprise one or more cationic lipids selected from the group consisting of DMTAP, DPTAP, DOTAP, DC-Chol, MoChol, HisChol, DPIM, CHIM, DORIE, DDAB, DAC-Chol, TC-Chol, DOTMA, DOGS, (C18) 2 Gly + N,N-dioctadecylamido-glycine, CTAP, CPyC, DODAP and DOEPC. 
     
     
         13 . The mixture of any of  claims 8  to  11 , wherein the lipid components comprise one or more cationic lipids selected from the group consisting of DOTAP, DC-Chol, MoChol and HisChol. 
     
     
         14 . The mixture of  claim 7  wherein the anionic lipid is CHEMS and the cationic lipid is MoChol. 
     
     
         15 . The mixture of  claim 7  wherein the anionic lipid is CHEMS and the cationic lipid is DOTAP. 
     
     
         16 . The mixture of  claim 7  wherein the anionic lipid is Cet-P and the cationic lipid is MoChol. 
     
     
         17 . The mixture of  claim 7  wherein the anionic lipid is DMGSucc and the cationic lipid is MoChol. 
     
     
         18 . The mixture of any of  claims 4  to  17 , wherein the lipid phase further comprises neutral lipids. 
     
     
         19 . The mixture of  claim 18  wherein the neutral lipids comprise sterols and derivatives thereof. 
     
     
         20 . The mixture of  claim 18  wherein the neutral lipids comprise neutral phospholipids. 
     
     
         21 . The mixture of  claim 20  wherein the neutral phospholipids comprise phosphatidylcholines. 
     
     
         22 . The mixture of  claim 21  wherein the phosphatidylcholines are selected from the group consisting of POPC, OPPC, natural or hydrogenated soy bean PC, natural or hydrogenated egg PC, DMPC, DPPC or DOPC and derivatives thereof. 
     
     
         23 . The mixture of  claim 22  wherein the phosphatidylcholine is POPC. 
     
     
         24 . The mixture of  claim 20  wherein the neutral phospholipids comprise mixtures of phosphatidylcholines and phosphoethanolamines. 
     
     
         25 . The mixture of  claim 24  wherein the phosphatidylcholines are selected from the group consisting of POPC, OPPC, natural or hydrogenated soy bean PC, natural or hydrogenated egg PC, DMPC, DPPC or DOPC and derivatives thereof and the phosphatidylethanolamines are selected from the group consisting of DOPE, DMPE, DPPE and derivatives thereof. 
     
     
         26 . The mixture of  claim 25  wherein the phosphatidylcholine is POPC and the phosphatidylethanolamine is DOPE. 
     
     
         27 . The mixture of  claim 18  wherein the neutral lipids are POPC and cholesterol. 
     
     
         28 . The mixture of  claim 24  wherein the amphoteric liposomes comprise DOPE, POPC, CHEMS and MoChol. 
     
     
         29 . The mixture of  claim 24  wherein the amphoteric liposome comprises POPC, DOPE, MoChol and DMGSucc. 
     
     
         30 . The mixture of  claim 27  wherein the amphoteric liposome comprises POPC, Chol, CHEMS and DOTAP. 
     
     
         31 . The mixture of  claim 27  wherein the amphoteric liposome comprises POPC, Chol, CetP and MoChol. 
     
     
         32 . A mixture comprising
 an amphoteric liposome comprising
 PNT-100 (SEQ ID NO:1251); 
 a mixture of lipid components with amphoteric properties wherein the mixture of lipid components comprises anionic and cationic components, wherein at least one such component is pH responsive and 
 neutral lipids, 
   
       wherein the neutral lipids comprise phosphatidylcholines, phosphatidylethanolamines or sterols and the mixture contains 30 to 70 mole % amphoteric lipids and 30 to 60 mole % neutral lipids. 
     
     
         33 . The mixture of  claim 32 , wherein the cationic lipids are selected from the group consisting of DMTAP, DPTAP, DPTAP, DOTAP, DC-Chol, TC-Chol, MoChol, HisChol, DPIM, CHIM, DORIE, DDAB, DAC-Chol, TC-Chol, DOTMA, DOGS, (C18) 2 Gly + N,N-dioctadecylamido-glycine, CTAB, CPyC, DODAP, DOEPC and derivatives thereof, and the anionic lipids are selected from the group consisting of DOGSucc, POGSucc, DMGSucc, DPGSucc, DGSucc, DMPS, DPPS, DOPS, POPS, DMPG, DPPG, DOPG, POPG, DMPA, DPPA, DOPA, POPA, CHEMS, Cet-P and derivatives thereof, and wherein the phosphatidylcholines are selected from the group consisting of POPC, OPPC, natural or hydrogenated soy bean PC, natural or hydrogenated egg PC, DMPC, DPPC or DOPC and derivatives thereof, the phosphatidylethanolamines are selected from the group consisting of DOPE, DMPE, DPPE and derivatives thereof, and the sterol is cholesterol or a derivative thereof. 
     
     
         34 . The mixture of  claim 32 , wherein the cationic lipids are selected from the group consisting of DOTAP, DC-Chol, MoChol and HisChol, and the anionic lipids are selected from the group consisting of DMGSucc, DOPA, CHEMS and Cet-P. 
     
     
         35 . The mixture of  claim 32 , wherein the cationic lipid is MoChol and the anionic lipid is CHEMS. 
     
     
         36 . The mixture of  claim 35 , wherein the mixture comprises 10 to 60 mole % of MoChol and 10 to 30 mole % of CHEMS. 
     
     
         37 . The mixture of  claim 32 , wherein the cationic lipid is DOTAP and the anionic lipid is CHEMS. 
     
     
         38 . The mixture of  claim 37 , wherein the mixture comprises 10 to 30 mole % of CHEMS and 5 to 40 mole % of DOTAP. 
     
     
         39 . The mixture of  claim 32 , wherein the cationic lipid is MoChol and the anionic lipid is Cet-P. 
     
     
         40 . The mixture of  claim 39 , wherein the mixture comprises 10 to 60 mole % of MoChol and 5 to 30 mole % of Cet-P. 
     
     
         41 . The mixture of  claim 32 , wherein the cationic lipid is MoChol and the anionic lipid is DMGSucc. 
     
     
         42 . The mixture of  claim 41 , wherein the mixture comprises 20 to 60 mole % of MoChol and 20 to 60 mole % of DMGSucc. 
     
     
         43 . The mixture of  claim 32 , wherein the neutral lipids comprise POPC and DOPE. 
     
     
         44 . The mixture of  claim 44 , wherein the mixture comprises 5 to 40 mole % of POPC and 20 to 50 mole % of DOPE. 
     
     
         45 . The mixture of  claim 32 , wherein the neutral lipids comprise POPC and Chol. 
     
     
         46 . The mixture of  claim 45 , wherein the mixture comprises 10 to 50 mole % of POPC and 30 to 50 mole % of Chol. 
     
     
         47 . The mixture of  claim 32 , wherein the amphoteric liposome comprises POPC, DOPE, MoChol and CHEMS. 
     
     
         48 . The mixture of  claim 47 , wherein the mixture comprises 3 to 20 mole % of POPC, 10 to 60 mole % of DOPE, 10 to 60 mole % of MoChol and 10 to 60 mole % of CHEMS. 
     
     
         49 . The mixture of  claim 47 , wherein the molar ratio of POPC/DOPE/MoChol/CHEMS is about 6/24/47/23. 
     
     
         50 . The mixture of  claim 47 , wherein the molar ratio of POPC/DOPE/MoChol/CHEMS is 15/45/20/20. 
     
     
         51 . The mixture of  claim 32 , wherein the amphoteric liposome comprises POPC, DOPE, DMGSucc and MoChol. 
     
     
         52 . The mixture of  claim 51 , wherein the mixture comprises 3 to 20 mole % of POPC, 10 to 40 mole % of DOPE, 15 to 60 mole % of DMGSucc and 15 to 60 mole % of MoChol. 
     
     
         53 . The mixture of  claim 52 , wherein the molar ratio of POPC/DOPE/DMGSucc/MoChol is about 6/24/23/47. 
     
     
         54 . The mixture of  claim 52 , wherein the molar ratio of POPC/DOPE/DMGSucc/MoChol is about 6/24/47/23. 
     
     
         55 . The mixture of  claim 32 , wherein the amphoteric liposome comprises POPC, Chol, CHEMS and DOTAP. 
     
     
         56 . The mixture of  claim 55 , wherein the mixture comprises 10 to 50 mole % of POPC, 20 to 60 mole % of Chol, 10 to 40 mole % of CHEMS and 5 to 20 mole % of DOTAP. 
     
     
         57 . The mixture of  claim 56 , wherein the molar ratio of POPC/Chol/CHEMS/DOTAP is about 30/40/20/10. 
     
     
         58 . The mixture of  claim 32 , wherein the amphoteric liposome comprises POPC, Chol, Cet-P and MoChol. 
     
     
         59 . The mixture of  claim 58 , wherein the mixture comprises 10 to 40 mole % of POPC, 20 to 50 mole % of Chol, 5 to 30 mole % of Cet-P and 10 to 40 mole % of MoChol. 
     
     
         60 . The mixture of  claim 59 , wherein the molar ratio of POPC/Chol/Cet-P/MoChol is about 35/35/10/20. 
     
     
         61 . The mixture of any one of the preceding claims wherein the amphoteric liposomes comprise a size between 50 and 500 ηm. 
     
     
         62 . The mixture of any of  claims 1 - 61  wherein the amphoteric liposome comprises a size between 80 and 300 ηm. 
     
     
         63 . The mixture of any of  claims 1 - 61  wherein the amphoteric liposome comprises a size between 90 and 200 ηm. 
     
     
         64 . The mixture of any of  claims 1 - 63  wherein the amphoteric liposome has a DNAi oligonucleotide concentration of at least 2 mg/ml at a lipid concentration of about 10 to 100 mM or less. 
     
     
         65 . The mixture of any of  claims 1 - 64  wherein the DNAi oligonucleotide comprises a DNAi oligomer that hybridizes under physiological conditions to nucleotides 500-2026 of SEQ ID NO:1249 or the complement thereof. 
     
     
         66 . The mixture of any of  claims 1 - 64  wherein the DNAi oligonucleotide comprises a DNAi oligomer that hybridizes under physiological conditions to nucleotides 500-1525 of SEQ ID NO:1249 or the complement thereof. 
     
     
         67 . The mixture of any of  claims 1 - 64  wherein the DNAi oligonucleotide comprises a DNAi oligomer that hybridizes under physiological conditions to nucleotides 800-1225 of SEQ ID NO:1249 or the complement thereof. 
     
     
         68 . The mixture of any of  claims 1 - 64  wherein the DNAi oligonucleotide comprises a DNAi oligomer that hybridizes under physiological conditions to nucleotides 900-1125 of SEQ ID NO:1249 or the complement thereof. 
     
     
         69 . The mixture of any of  claims 1 - 64  wherein the DNAi oligonucleotide comprises a DNAi oligomer that hybridizes under physiological conditions to nucleotides 950-1075 of SEQ ID NO:1249 or the complement thereof. 
     
     
         70 . The mixture of any of  claims 1 - 64  wherein the DNAi oligonucleotide comprises a DNAi oligomer that hybridizes under physiological conditions to nucleotides 970-1045 of SEQ ID NO:1249 or the complement thereof. 
     
     
         71 . The mixture of any of  claims 1 - 64  wherein the DNAi oligonucleotide comprises a DNAi oligomer selected from the group consisting of SEQ ID NOs:1250, 1251, 1252, 1253, 1267-1447 and the complements thereof. 
     
     
         72 . The mixture of any of  claims 1 - 64  wherein the DNAi oligonucleotide comprises a DNAi oligomer selected from the group consisting of SEQ ID NOs 1250, 1251, 1267, 1268, 1276, 1277, 1285, 1286 and the complements thereof. 
     
     
         73 . The mixture of any of  claims 1 - 64  wherein the DNAi oligonucleotide comprises a DNAi oligomer selected from the group consisting of SEQ ID NOs 1250, 1251, 1289-1358 and the complements thereof. 
     
     
         74 . The mixture of any of  claims 1 - 64 , wherein the DNAi oligonucleotide comprises SEQ ID NO:1250 or 1251. 
     
     
         75 . A method comprising
 (a) providing the mixture of any of  claims 1 - 64  and a cell or animal capable of expressing the bcl-2 gene, and   (b) introducing the mixture into the cell or animal.   
     
     
         76 . The method of  claim 75 , wherein introducing the composition results in a reduction of proliferation of the cell, or induces cell death. 
     
     
         77 . The method of  claim 75 , wherein the cell is a cancer cell. 
     
     
         78 . The method of  claim 75 , wherein the animal is a non-human animal. 
     
     
         79 . The method of  claim 75 , wherein the animal is a human. 
     
     
         80 . The method of  claim 78 , wherein the mixture is introduced to the animal at a dosage of between 0.01 mg to 100 mg per kg of body weight. 
     
     
         81 . The method of  claim 78 , wherein the mixture is introduced to the animal one or more times per day. 
     
     
         82 . The method of  claim 78 , wherein the mixture is introduced to the animal continuously. 
     
     
         83 . The method of  claim 78 , wherein the mixture is introduced to the animal by one or more routes of administration selected from the group consisting of topical, pulmonary, intraocular, intranasal, parenteral, and a medical device. 
     
     
         84 . The method of  claim 74 , wherein the cell is in cell culture. 
     
     
         85 . The method of  claim 74 , further comprising the step of introducing a test compound to the cell or animal. 
     
     
         86 . The method of  claim 85 , wherein the test compound is a chemotherapy agent. 
     
     
         87 . The method of  claim 86 , wherein animal has cancer which is selected from the group consisting of pancreatic cancer, colon cancer, breast cancer, bladder cancer, lung cancer, leukemia, prostate cancer, lymphoma, ovarian cancer and melanoma. 
     
     
         88 . A pharmaceutical composition comprising the mixture of any of  claims 1  to  74 . 
     
     
         89 . A mixture comprising an amphoteric liposome and a DNAi oligonucleotide comprising SEQ ID NO:1251 or 1250 wherein the liposome comprises POPC, DOPE, MoChol and CHEMS in the molar ratio of POPC/DOPE/MoChol/CHEMS of 15/45/20/20. 
     
     
         90 . A mixture comprising amphoteric liposome and a DNAi oligonucleotide comprising SEQ ID NO:1251 or 1250 wherein the liposome comprises POPC, DOPE, MoChol and CHEMS in the molar ratio of POPC/DOPE/MoChol/CHEMS of 6/24/47/23.