Peptide/Lipid Complex Formation by Co-Lyphilization

Abstract

The invention relates to the formation of peptide/lipid vesicles and complexes through the co-lyophilization of peptides, preferably that are able to adopt an amphipathic alphahelical conformation, and one or more lipids. A single solution which solubilizes both the peptides and lipids or two separate solutions may be lyophilized.

1 . A method of preparing a lyophilized peptide/lipid product which comprises co-lyophilizing one or more peptides, which are able to adopt an amphipathic conformation, or peptide analogues, and one or more lipids in a solvent system to form a peptide/lipid product, wherein said product can be rehydrated to form peptide/lipid complexes.  
     
     
         2 . The method of  claim 1  wherein said peptide is a lipid binding protein.  
     
     
         3 . The method of  claim 1  wherein said peptide analogue is an analogue of ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoE or another apoprotein.  
     
     
         4 . The method of  claim 1  wherein said peptide is a protein.  
     
     
         5 . The method of  claim 1  wherein said lipid is a natural lipid, synthetic lipid, saturated lipid, unsaturated lipid or mixtures thereof.  
     
     
         6 . The method of  claim 5  wherein said lipid is selected from the group consisting of egg phosphatidylcholine, soybean phosphatidylcholine, ether phospholipids, small alkyl chain phospholipids, cholesterol, cholesterol derivatives, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine dioleophosphatidylethanolamine, dilauroylphosphatidylglycerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin sphingolipids, phosphatidylglycerol, diphosphatidylglycerol dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, phosphatidic acid, galactocerebroside, gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride, aminophenylglycoside, and 3-cholesteryl-6′-(glycosylthio)hexyl ether glycolipids, and mixtures thereof.  
     
     
         7 . The method of  claim 1  which further comprises sterilizing said product prior to, during or after lyophilization.  
     
     
         8 . The method of  claim 1  wherein said peptide/lipid complex is sterile.  
     
     
         9 . The method of  claim 1  further comprising aliquoting a solution of said peptide and said lipid into individual containers before lyophilization to form a single unit dosage form.  
     
     
         10 . A pharmaceutical unit dosage form which comprises a sterile lyophilized peptide/lipid mixture prepared according to  claim 1  or  7 .  
     
     
         11 . A method of preparing a lyophilized peptide/lipid product which comprises (a) solubilizing at least one amphipathic peptide or peptide analogue in a first solution, (b) solubilizing at least one lipid in a second solution, wherein said second solution is miscible with said first solution, (c) combining said first solution with said second solution to form a peptide/lipid solution, and (d) lyophilizing said peptide/lipid solution so that lyophilized peptide/lipid product is formed which can be rehydrated to form peptide/lipid complexes.  
     
     
         12 . The method of  claim 11  wherein said peptide is a lipid binding protein.  
     
     
         13 . The method of  claim 1  wherein said peptide is a protein.  
     
     
         14 . The method of  claim 9  wherein said peptide analogue is an ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoE or another apoprotein analogue.  
     
     
         15 . The method of  claim 11  wherein said lipid is a natural lipid, a synthetic lipid, a saturated lipid, unsaturated lipid or mixtures thereof.  
     
     
         16 . The method of  claim 15  wherein said lipid is selected from the group consisting of egg phosphatidylcholine, cholesterol, cholesterol derivatives, ether phospholipidsr soybean phosphatidycholine, small alkyl chain phospholipids, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine dioleophosphatidylethanolamine, dilauroylphosphatidylglycerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin sphingolipids, phosphatidylglycerol, diphosphatidylglycerol dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, phosphatidic acid, galactocerebroside, gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride, aminophenylglycoside, and 3-cholesteryl-6′-(glycosylthio)hexyl ether glycolipids, and mixtures thereof.  
     
     
         17 . The method of  claim 11  wherein said peptide/lipid solution is sterile.  
     
     
         18 . The method of  claim 11  where said peptide/lipid complex is sterile.  
     
     
         19 . The method of claims  11 , further comprising aliquoting said peptide/lipid solution into individual containers before lyophilization to form a single unit dosage form.  
     
     
         20 . The method of  claim 11  which further comprises a sterilization step prior to, during or after lyophilization.  
     
     
         21 . A pharmaceutical unit dosage form which comprises a sterile and stable lyophilized peptide/lipid mixture prepared according to  claim 11  or  19 .  
     
     
         22 . A peptide/lipid complexes formed by the process comprising lyophilizing one or more amphipathic peptides or peptide analogues and at least one lipid in a solvent system to form a dehydrated peptide/lipid product which can be rehydrated to form peptide/lipid complexes.  
     
     
         23 . The peptide/lipid complexes of  claim 22  wherein said peptide is a lipid binding protein.  
     
     
         24 . The peptide/lipid complexes of  claim 22  wherein said peptide is an ApoA1 analog.  
     
     
         25 . The peptide/lipid complex of  claim 22  wherein said lipid is a natural, synthetic, saturated, unsaturated lipid or mixtures thereof.  
     
     
         26 . The peptide/lipid complexes of  claim 25  wherein said lipid is selected from the group consisting of egg phosphatidylcholine, soybean phosphatidycholine, cholesterol, cholesterol derivatives, small alkyl chain phospholipids, ether phospholipids, dipalmitoylphosphatidylcholine, dinyristoylphosphatidylcholine, distearoylphosphatidylcholine 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine dioleophosphatidylethanolamine, dilauroylphosphatidylglycerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin sphingolipids, phosphatidylglycerol, diphosphatidylglycerol dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, phosphatidic acid, galactocerebroside, gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride, aminophenylglycoside, and 3-cholesteryl-6t-(glycosylthio)hexyl ether glycolipids, and mixtures thereof.  
     
     
         27 . The peptide/lipid complexes of  claim 22  wherein said complex is sterile.  
     
     
         28 . The peptide/lipid complexes of  claim 22 , wherein said complex is formulated into sterile unit dosage.  
     
     
         29 . A sterile, lyophilized composition comprising a sterile preparation of a complex formed between a peptide which can adopt a amphipathic alpha helical conformation, or a peptide analogue and a lipid.  
     
     
         30 . The composition of  claim 28  wherein said preparation is provided in a sterile unit dosage formulation.  
     
     
         31 . A lyophilized composition which comprises a peptide/lipid complex wherein the peptide is a lipid binding protein or an ApoA1, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoE or another apoprotein analogue.  
     
     
         32 . The lyophilized composition of  claim 31  wherein the peptide/lipid complex is a vesicle, micelle, liposome, discoidal particle, spherical particle, or mixture thereof.  
     
     
         33 . A lyophilized composition which comprises a peptide/lipid complex wherein said peptide can adopt an amphipathic alpha helical conformation.  
     
     
         34 . The lyophilized composition of  claim 33  wherein the peptide/lipid complex is a vesicle, micelle, liposome, discoidal particle, spherical particle, or mixture thereof.  
     
     
         35 . The composition of claims  31  or  33  wherein said composition is sterile.  
     
     
         36 . The composition of  claim 29  or  31  said analogue is not a peptide or a protein.  
     
     
         37 . A method of preparing a lyophilized peptide/lipid roduct which comprises: 
 co-lyophilizing one or more peptides or peptides analogues, which are able to adopt an amphipathic conformation, with one or more lipids in a ratio of peptide to lipid of from about 2 to about 200 in a solvent system for an amount of time sufficient to form a peptide/lipid product which can be rehydrated to form peptide/lipid complexes in solution.