Fusogenic properties of saposin c and related proteins and peptides for application to transmembrane drug delivery systems

Abstract

The present invention comprises a method for delivering pharmaceutical and/or imaging agents within and/or through the dermal, mucosal and other cellular membranes, and across the blood-brain barrier, utilizing a fusogenic protein. The fusogenic protein is associated with a phospholipid membrane, such as a liposome. The liposome may include dioleoylphosphatidylserine, a negatively charged long-chain lipid. Alternatively, the liposome is comprised of a mixture of negatively charged long-chain lipids, neutral long-chain lipids, and neutral short-chain lipids. Preferred fusogenic proteins include saposin C and other proteins, polypeptides and peptide analogs derived from saposin C. The active agent contained within the liposome may comprise biomolecules and/or organic molecules. This technology can be used for both cosmetic and medicinal applications in which the objective is delivery of the active agent within and/or beneath biological membranes or across the blood-brain barrier and neuronal membranes.

What is claimed is: 
     
         1 . A method for treating a central nervous system (CNS) disorder comprising delivering an agent through a targeted biological membrane, wherein the method comprises applying to the membrane a composition comprising:
 a) one or more phospholipid selected from the group consisting of long-chain phospholipids, short-chain lipids and mixtures thereof;   b) a safe and effective amount of an agent;   c) a fusogenic protein or polypeptide derived from prosaposin; and   d) a pharmaceutically acceptable carrier,   wherein the concentration of the fusogenic protein or polypeptide is of a sufficient amount to deliver the agent through the membrane, and wherein the composition forms a liposome capable of fusing with the targeted biological membrane;   and wherein the CNS disorder is selected from the group consisting of: neuroblastoma, cerebral inflammation, metachromatic leukodystrophy (MLD), Niemann-Pick, stroke, Parkinson's, Alzheimer's diseases, demyelination disorders, retinal neuropathy, Huntington's disease, A.L.S., multiple sclerosis, neuro-AIDS, brain or spinal cord trauma, autism, lysosomal storage disorders, fragile X syndrome, inherited ataxias, and blindness.   
     
     
         2 . The method according to  claim 1  wherein the phospholipids are a mixture of anionic long-chain lipids and neutral long-chain lipids. 
     
     
         3 . The method according to  claim 1  wherein the phospholipids are a mixture of anionic long-chain lipids, neutral long-chain lipids and neutral short-chain lipids. 
     
     
         4 . The method of  claim 3  wherein the liposome contains cationic phospholipids. 
     
     
         5 . The method of  claim 3  wherein the amounts of anionic long-chain lipid, neutral long-chain lipid and short-chain lipid is governed by the formula ([neutral long chain lipid]+[anionic long chain lipid))/(short chain lipid) equals about 4. 
     
     
         6 .- 9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the pH of the composition is between about 6.8 and 2. 
     
     
         11 . The method of  claim 10 , herein the pH of the composition is between about 5.5 and about 2. 
     
     
         12 . The method of  claim 11  comprising the further step of neutralizing the pH with a neutralizing buffer. 
     
     
         13 . The method of  claim 12 , wherein the pH of the composition is adjusted such that it form liposomes having mean diameters of between 50 and 350 nanometers. 
     
     
         14 . The method of  claim 12 , wherein the pH of the composition is adjusted such that it form liposomes having mean diameters of about 200 nanometers. 
     
     
         15 .- 28 . (canceled) 
     
     
         29 . The method of  claim 1  wherein the fusogenic protein or polypeptide is selected from the group consisting of saposin A, saposin C, SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ ID Nos. 3, and mixtures thereof. 
     
     
         30 . The method of  claim 1  wherein administration of the composition is selected from a transdermal patch, enterally, trans-nasally, intravenously, intramuscularly or topically. 
     
     
         31 .- 36 . (canceled) 
     
     
         37 . A method for treating a CNS disorder comprising delivering a pharmaceutical or imaging agent across a membrane of the blood-brain barrier wherein the method comprises the administration to the membrane of a composition comprising:
 a. phospholipids selected from the group consisting of anionic long-chain phospholipids, neutral long chain lipids, neutral short chain lipids, anionic short-chain lipids and mixtures thereof;   b. a safe and effective amount of the pharmaceutical agent or imaging agent;   c. a fusogenic protein or polypeptide derived from prosaposin; and   d. a pharmaceutically acceptable carrier,   wherein the concentration of the liposomes are of a sufficient amount to deliver a safe and effective amount of the pharmaceutical agent through the membrane and wherein the phospholipids form a liposome having an overall negative charge;   and wherein the CNS disorder is selected from the group consisting of: neuroblastoma, cerebral inflammation, metachromatic leukodystrophy (MLD), Niemann-Pick, stroke, Parkinson's, Alzheimer's diseases, demyelination disorders, retinal neuropathy, Huntington's disease, A.L.S., multiple sclerosis, neuro-AIDS, brain or spinal cord trauma, autism, lysosomal storage disorders, fragile X syndrome, inherited ataxias, and blindness.   
     
     
         38 . The method according to  claim 37  wherein the phospholipids are a mixture of anionic long-chain lipids and neutral long-chain lipids. 
     
     
         39 . The method according to  claim 37  wherein the phospholipids are a mixture of anionic long-chain lipids, neutral long-chain lipids and neutral short-chain lipids. 
     
     
         40 . The method of  claim 39  wherein the liposome contains cationic phospholipids. 
     
     
         41 - 42 . (canceled) 
     
     
         42 . The method according to  claim 37  wherein the pH of the composition is between about 5.5 and about 2. 
     
     
         43 . The method according to  claim 37  wherein the fusogenic protein or polypeptide is saposin C. 
     
     
         45 - 50 . (canceled) 
     
     
         51 . A method for treating Gaucher's Disease wherein the method comprises the administration of a composition to a subject in need of such therapy comprising:
 anionic liposomes containing one or more phospholipid selected from the group consisting of anionic long chain phospholipids, neutral long chain phospholipids, neutral short chain phospholipids, anionic short chain phospholipids and mixtures thereof in which the amounts of anionic long-chain lipid, neutral long-chain lipid   and short-chain lipid is governed by the formula ([neutral long chain lipid]+[anionic long chain lipid])/(neutral short chain lipid) equals about 4;   a safe and effective amount of acid beta-glucosidase; a fusogenic protein or polypeptide derived from prosaposin; and   a pharmaceutically acceptable carrier, wherein the pH of the composition between about 5.5 and about 2, the concentration of the fusogenic protein or polypeptide is of a sufficient amount to deliver the pharmaceutical agent through the membrane and the saposin C is associated with the surface of the liposome through an electrostatic and hydrophobic interaction.   
     
     
         52 . The method of  claim 51  wherein the concentration of the liposome is in at least about a 1 to about 10-fold excess, by molar ratio, to that of fusogenic protein or polypeptide.