Encapsulation of Plasmid DNA (Lipogenes) and Therapeutic Agents with Nuclear Localization Signal/Fusogenic Peptide Conjugates into Targeted Liposome Complexes

Abstract

A method is disclosed for encapsulating plasmids, oligonucleotides or negatively-charged drugs into liposomes having a different lipid composition between their inner and outer membrane bilayers and able to reach primary tumors and their metastases after intravenous injection to animals and humans. The formulation method includes complex formation between DNA with cationic lipid molecules and fusogenic/NLS peptide conjugates composed of a hydrophobic chain of about 10-20 amino acids and also containing four or more histidine residues or NLS at their one end. The encapsulated molecules display therapeutic efficacy in eradicating a variety of solid human tumors including but not limited to breast carcinoma and prostate carcinoma. Combination of the plasmids, oligonucleotides or negatively-charged drugs with other anti-neoplastic drugs (the positively-charged cis-platin, doxorubicin) encapsulated into liposomes are of therapeutic value. Also of therapeutic value in cancer eradication are combinations of encapsulated the plasmids, oligonucleotides or negatively-charged drugs with HSV-tk plus encapsulated ganciclovir.

1 . A method for producing micelles with entrapped therapeutic agents, comprising:
 a) combining an effective amount of a negatively charged therapeutic agent with an effective amount of a cationic lipid in a ratio where about 30% to about 90% the negatively charged atoms are neutralized by positive charges on lipid molecules to form an electrostatic micelle complex in about 20% to about 80% ethanol; and   b) combining the micelle complex of step a) with an effective amount of a fusogenic-karyophilic peptide conjugates in a ratio range of about 0.0 to about 0.3, thereby producing micelles with entrapped therapeutic agents.   
     
     
         2 . The method of  claim 1 , wherein the negatively charged therapeutic agent is a therapeutic agent selected from the group consisting of a polynucleotide and a negatively charged drug. 
     
     
         3 . The method of  claim 2 , wherein the polynucleotide is a DNA polynucleotide or an RNA polynucleotide. 
     
     
         4 . The method of  claim 2 , wherein the polynucleotide is a DNA polynucleotide. 
     
     
         5 . The method of  claim 4 , wherein the DNA polynucleotide comprises plasmid DNA. 
     
     
         6 . The method of  claim 1 , further comprising combining an effective amount of an anionic lipid in step a). 
     
     
         7 . The method of  claim 6 , wherein the anionic lipid is dipalmitoyl phosphatidyl glycerol (DDPG) or a derivative thereof. 
     
     
         8 . The method of  claim 4 , further comprising combining an effective amount of a DNA condensing agent selected from the group consisting of spermine, spermidine, polylysine, polyarginine, polyhistidine, polyornithine and magnesium or a divalent metal ion. 
     
     
         9 . The method of  claim 5 , wherein the plasmid DNA comprises a sequence encoding p53, HSV-tk, p21, Bax, Bad, IL-2, IL-12, GM-CSF, angiostatin, endostatin and oncostatin. 
     
     
         10 . The method of  claim 1 , wherein the cationic lipids are selected from the group consisting of 3β-(N—(N′,N′-dimethylaminoethane)carbamoyl)cholesterol, dimethyldioctadecyl ammonium bromide (DDAB), N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide (DMRIE), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), dioctadecylamidoglycylspermine (DOGS), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dipalmitoyl-3-trimethylammonium propane (DPTAP), 1,2-disteroyl-3-trimethylammonium propane (DSTAP). 
     
     
         11 . The method of  claim 10 , wherein the cationic lipids are combined with the fusogenic lipid DOPE in a molar ratio from about 1:1 to about 2:1. 
     
     
         12 . The method of  claim 11 , wherein the cationic lipids are combined with the fusogenic lipid DOPE in a molar ratio of 1:1. 
     
     
         13 . The method of  claim 1 , wherein the fusogenic-karyophilic peptide is an NLS peptide. 
     
     
         14 . The method of  claim 13 , wherein the NLS peptide is a peptide selected from the group consisting of Seq. ID Nos. 20-622. 
     
     
         15 . The method of  claim 1 , wherein the fusogenic-karyophilic peptide conjugate is a sole fusogenic peptide. 
     
     
         16 . The method of  claim 1 , wherein the NLS peptide component of the fusogenic-karyophilic peptide conjugate is an NLS peptide selected from the group consisting of Seq. ID Nos. 20-622. 
     
     
         17 . The method of  claim 1 , wherein the fusogenic/NLS peptide conjugates comprise amino acid sequences selected from the group consisting of (KAWLKAF) 3  (SEQ ID NO:1), GLFKAAAKLLKSLWKLLLKA (SEQ ID NO:2), LLLKAFAKLLKSLWKLLLKA (SEQ ID NO:3) as well as all derivatives of the prototype (Hydrophobic 3 Karyophilic 1 Hydrophobic 2 Karyophilic 1 ) 2-3  where Hydrophobic is any of the A, I, L, V, P, G, W, F and Karyophilic is any of the K, R, or H, containing a positively-charged residue every 3rd or 4th amino acid, that form alpha helices and direct a net positive charge to the same direction of the helix. 
     
     
         18 . The method of  claim 1 , wherein the fusogenic/NLS peptide conjugate comprise an amino acid sequence selected from the group consisting of GLFKAIAGFIKNGWKGMIDGGGYC (SEQ ID NO:4) from influenza virus hemagglutinin HA-2 and YGRKKRRQRRR (SEQ ID NO:5) from TAT of HIV. 
     
     
         19 . The method of  claim 1 , wherein the fusogenic/NLS peptide conjugate comprise an amino acid sequence selected from the group consisting of MSGTFGGILAGLIGLL(K/R/H) 1-6  (SEQ ID NO:6), derived from the N-terminal region of the S protein of duck hepatitis B virus but with the addition of one to six positively-charged lysine, arginine or histidine residues, and combinations of these, GAAIGLAWIPYFGPAA (SEQ ID NO:7) derived from the fusogenic peptide of the Ebola virus transmembrane protein; residues 53-70 (C-terminal helix) of apolipoprotein (apo) AII peptide, the 23-residue fusogenic N-terminal peptide of HIV-1 transmembrane glycoprotein gp41, the 29-42-residue fragment from Alzheimer's beta-amyloid peptide, the fusion peptide and N-terminal heptad repeat of Sendai virus, the 56-68 helical segment of lecithin cholesterol acyltransferase. 
     
     
         20 . The method of any of  claim 13  to  19 , wherein the NLS peptide component in fusogenic/NLS peptide conjugates are synthetic peptides containing the above said NLS but further modified by additional K, R, H residues at the central part of the peptide or with P or G at the N- or C-terminus. 
     
     
         21 . The method of  claim 13 , wherein the fusogenic peptide/NLS peptide conjugates are linked to each other with a short amino acid stretch representing an endogenous protease cleavage site. 
     
     
         22 . The method of  claim 1 , wherein the structure of the preferred prototype fusogenic/NLS peptide conjugate used in this invention is: PKKRRGPSP(L/A/I) 12-20  (SEQ ID NO:8) where (L/A/I) 12-20  is a stretch of 12-20 hydrophobic amino acids containing A, L, I, Y, W, F and other hydrophobic amino acids. 
     
     
         23 . The method of  claim 1 , wherein the fusogenic/NLS peptide conjugates are added to the mixture of DNA/cationic lipid and are incorporated into micelles. 
     
     
         24 . The method of  claim 1 , further comprising combining an effective amount of an encapsulating lipid solution to step b). 
     
     
         25 . The method of  claim 24 , wherein the encapsulating lipid is a lipid comprising cholesterol (40%), dioleoylphosphatidylethanolamine (DOPE) (20%), palmitoyloleoylphosphatidylcholine (POPC) (12%), hydrogenated soy phosphatidylcholine (HSPC) (10%), distearoylphosphatidylethanolamine (DSPE) (10%), sphingomyelin (SM) (5%), and derivatized vesicle-forming lipid M-PEG-DSPE (3%). 
     
     
         26 . The method of  claim 24 , wherein the encapsulating lipid is a liposome. 
     
     
         27 . The method of  claim 26 , wherein the liposomes comprises vesicle-forming lipids and between about 1 to about 7 mole percent of distearoylphosphatidyl ethanolamine (DSPE) derivatized with an effective amount of polyethyleneglycol. 
     
     
         28 . The method of  claim 27 , wherein the liposomes have a selected average size of about 80 to about 160 nm. 
     
     
         29 . The method of  claim 27 , wherein the polyethyleneglycol has a molecular weight from about 1,000 to about 5,000 daltons. 
     
     
         30 . A micelle with an entrapped therapeutic agent produced by the method of  claim 1 . 
     
     
         31 . A liposome encapsulated therapeutic agent produced by the method of  claim 24 . 
     
     
         32 . The method of  claim 31 , wherein the therapeutic agent further comprises regulation by a liver, spleen or bone marrow regulatory DNA sequence. 
     
     
         33 . The method of  claim 32 , wherein the regulatory DNA sequence is nuclear matrix DNA isolated from liver, spleen or bone marrow cells. 
     
     
         34 . A method for delivering a therapeutic agent in vivo, comprising administration of an effective amount of the micelle of  claim 30  to a subject. 
     
     
         35 . The method of  claim 34 , wherein the therapeutic agent further comprises regulation by a tumor-specific regulatory DNA sequence. 
     
     
         36 . The method of  claim 35 , wherein the tumor-specific regulatory sequence is nuclear matrix DNA isolated from specific tumor cells. 
     
     
         37 . A method for delivering a therapeutic agent in vivo, comprising administration of an effective amount of the liposome encapsulated agent of  claim 31  to the subject. 
     
     
         38 . The method of  claim 34  or  37 , wherein the administration is intravenous administration or by injection. 
     
     
         39 . A micelle with an entrapped DNA polynucleotide produced by the method of  claim 9 . 
     
     
         40 . A method for reducing tumor size in a subject comprising administration of an effective amount of the micelle of  claim 39  to the subject. 
     
     
         41 . The method of  claim 40 , further comprising administration of an effective amount of a second therapeutic agent, wherein the agent is selected from the group consisting of ganciclovir, 5-fluorocytosine, an antisense oligonucleotides a ribozyme, and a triplex-forming oligonucleotide directed against genes that control the cell cycle or signaling pathways. 
     
     
         42 . The method of  claim 41 , further comprising administration of an effective amount of a second therapeutic agent, wherein the second therapeutic agent is selected from the group consisting of adriamycin, angiostatin, azathioprine, bleomycin, busulfane, camptothecin, carboplatin, carmustine, chlorambucile, chlormethamine, chloroquinoxaline sulfonamide, cisplatin, cyclophosphamide, cycloplatam, cytarabine, dacarbazine, dactinomycin, daunorubicin, didox, doxorubicin, endostatin, enloplatin, estramustine, etoposide, extramustinephosphat, flucytosine, fluorodeoxyuridine, fluorouracil, gallium nitrate, hydroxyurea, idoxuridine, interferons, interleukins, leuprolide, lobaplatin, lomustine, mannomustine, mechlorethamine, mechlorethaminoxide, melphalan, mercaptopurine, methotrexate, mithramycin, mitobronitole, mitomycin, mycophenolic acid, nocodazole, oncostatin, oxaliplatin, paclitaxel, pentamustine, platinum-triamine complex, plicamycin, prednisolone, prednisone, procarbazine, protein kinase C inhibitors, puromycine, semustine, signal transduction inhibitors, spiroplatin, streptozotocine, stromelysin inhibitors, taxol, tegafur, telomerase inhibitors, teniposide, thalidomide, thiamiprine, thioguanine, thiotepa, tiamiprine, tretamine, triaziquone, trifosfamide, tyrosine kinase inhibitors, uramustine, vidarabine, vinblastine, vinca alcaloids, vincristine, vindesine, vorozole, zeniplatin, zeniplatin, and zinostatin.