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WO2007065017A2 - Systeme d'administration de liposomes cationiques a oligonucleotides - Google Patents

Systeme d'administration de liposomes cationiques a oligonucleotides Download PDF

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Publication number
WO2007065017A2
WO2007065017A2 PCT/US2006/046298 US2006046298W WO2007065017A2 WO 2007065017 A2 WO2007065017 A2 WO 2007065017A2 US 2006046298 W US2006046298 W US 2006046298W WO 2007065017 A2 WO2007065017 A2 WO 2007065017A2
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WIPO (PCT)
Prior art keywords
mixture
oligomer
seq
cholesterol
acid
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PCT/US2006/046298
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WO2007065017A3 (fr
Inventor
Neal Clifford Goodwin
Viktor Peykov
Wendi Rodrigueza
Sydney Ugwu
Tong Xuan
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Sierra Oncology Inc
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ProNAi Therapeutics Inc
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Publication of WO2007065017A2 publication Critical patent/WO2007065017A2/fr
Publication of WO2007065017A3 publication Critical patent/WO2007065017A3/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers

Definitions

  • the invention relates to compositions and methods of using the same to treat cancer.
  • the invention provides oligonucleotides sequestered with cationic liposomes for the treatment of cancer.
  • Oncogenes have become the central concept in understanding cancer biology and may provide valuable targets for therapeutic drugs.
  • oncogenes are overexpressed, and may be associated with tumorigenicity (Tsujimoto et al, Science 228:1440-1443 [1985]).
  • tumorigenicity Tujimoto et al, Science 228:1440-1443 [1985]
  • high levels of expression of the human bcl-2 gene have been found in all lymphomas with a t(14; 18) chromosomal translocations including most follicular B cell lymphomas and many large cell non-Hodgkin's lymphomas.
  • oncogenes include TGF- ⁇ , c-ki-ras, ras, Her-2, and c-myc. [004] The expression of oncogenes may be inhibited by single stranded oligonucleotides. Nucleic acid therapeutics, however, often lack therapeutic efficacy due to instability in body fluids or inefficient uptake into cells.
  • the invention provides therapeutic compositions and methods for preparing and using liposomes for the delivery of oligonucleotides for treating cancer.
  • the invention provides a mixture of liposomes and oligonucleotides that alter the expression of genes involved in cancer and/or alter cell growth.
  • the mixture includes a plurality of liposomes including a cationic colipid, a zwitterionic colipid, an oligomer, cholesterol, a stabilizer (e.g., tocopherol acid succinate), and optionally an isotonicity adjuster (e.g., sucrose, trehalose, maltose, lactose, or glucose).
  • the mixture may have a charge ratio from about 1.5 to about 2.5 (e.g., about 2.0).
  • the oligomer contained in the mixture can include the sequence of PNT-100 (SEQ ID NO:1250 or 1251).
  • the mixture may include about 2 mg/mL of PNT-100 oligomer.
  • the concentration of total lipids in the mixture ranges from about 24.0 mg/ml to 36.0 mg/mL.
  • the mixture comprises from about 3.0 mg/mL to about 6.0 mg/mL of cholesterol.
  • the zwitterionic colipid contained in the mixture includes DOPC, e.g., in the concentration from about 10 mg/mL to about 24 mg/mL of DOPC.
  • the cationic colipid contained in the mixture includes (R)-PCL-2, e.g., in the contraction from about 6.3 mg/mL to about 10.6 mg/mL.
  • the mixture has a pH of between about 5.1 and about 7.9.
  • the mixture comprises from about 14.9 mg/mL to about 18.9 mg/mL of DOPC, from about 4.4 mg/mL to about 4.8 mg/mL of cholesterol, from about 6.3 mg/mL to about 10.6 mg/mL of (R)- PCL-2, tocopherol acid succinate, and sucrose.
  • the molar ratio of total lipids to oligomer in the mixture is from about 100 to about 180 (e.g., about 144).
  • the invention provides a liposomal mixture comprising a zwitterionic colipid, a sterol, a bcl-2 oligomer and a cationic cardiolipin analog with the general structure I.
  • n is 1 or 2;
  • Z 1 , Z 2 , Z 3 , and Z 4 are independently -O-C(O)-, -O-, -S-, or - NH-C(O)-;
  • R 1 , R 2 , R 3 , and R 4 are independently H, C 1 to C 32 saturated or unsaturated aliphatic, optionally substituted with one or more hydroxy, amino, sulfinyl, alkoxy, cycloaliphatic, PEG, halo, or combinations thereof;
  • R 5 is H, aliphatic, alkoxy, cycloaliphatic, alkanoyl, alkenoyl, or alkynoyl, each optionally substituted with one or more hydroxy, amino, sulfinyl, epoxy, cycloalkyl, PEG, halo, or combinations thereof; an amino acid, a peptide, a peptidomimetic moiety, a dipeptide, a polypeptid
  • R 12 , R 13 and R 14 are independently H, aliphatic, alkoxy, cycloaliphatic, alkanoyl, alkenoyl, or alkynoyl, each optionally substituted with one or more hydroxy, amino, sulfinyl, alkoxy, cycloaliphatic, PEG, halo, or combinations thereof; an amino acid, a peptide, a peptidoniimetic moiety, a dipeptide, a polypeptide, a protein, an oligosaccharide or polysaccharide, a polyamine, a heterocyclic, a nucleoside or a polynucleotide; and X is a non-toxic anion.
  • n is 1,
  • a and A' are independently selected from the
  • R 6 and R 7 can be the same or different, and can independently be absent or comprise a linker comprising a C 1 to C 32 alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl or an alkyloxy or substituted alkyloxy group such as a PEGylated ether containing from 1 to 500 PEG (polyethylene glycol units.
  • the R 8 groups are the same or different and are independently C 1 to C 25 saturated or unsaturated alkyl, alkyloxy, substituted alkyl or substituted alkyloxy.
  • R 9 and R 10 are independently absent or comprise a linker comprising a C 1 to C 32 aliphatic, cycloaliphatic, or alkoxy, each optionally substituted with one or more hydroxy, amino, sulfinyl, alkoxy, cycloaliphatic, PEG, halo, or combinations thereof.
  • B is either a non-
  • R 11 is a C 2 to C 32 alkylene, alkenylene, or alkoxo, each optionally substituted with one or more hydroxy, amino, sulfinyl, alkoxy, cycloaliphatic, PEG, halo, or combinations thereof.
  • R 12 , Ri 3 and Ri 4 are independently H, aliphatic, alkoxy, cycloaliphatic, alkanoyl, alkenoyl, or alkynoyl, each optionally substituted with one or more hydroxy, amino, sulfinyl, alkoxy, cycloaliphatic, PEG, halo, or combinations thereof; an amino acid, a peptide, a peptidomimetic moiety, a dipeptide, a polypeptide, a protein, an oligosaccharide or polysaccharide, a polyamine, a heterocyclic, a nucleoside or a polynucleotide.
  • X is a non-toxic anion.
  • n is 2, A and A' can independently be structures II and III.
  • Z 1 to Z 4 can be the same or different and can be independently -O-C(O)-, -O-, -S- or -NH-C(O)-.
  • R 1 , R 2 , R 3 , and R 4 are the same or different and can be independently H, C 1 to C 32 saturated or unsaturated alkyl, alkenyl, or alkynyl groups, optionally hydroxylated, aminenated, thiolated epoxylated, cyclolated, PEGylated, halogenated, or substituted with combinations thereof.
  • R 5 can be H, aliphatic, alkoxy, cycloaliphatic, alkanoyl, alkenoyl, or alkynoyl, each optionally substituted with one or more hydroxy, amino, sulfinyl, epoxy, cycloalkyl, PEG, halo, or combinations thereof; an amino acid, a peptide, a peptidomimetic moiety, a dipeptide, a polypeptide, a protein, an oligosaccharide or polysaccharide, a polyamine, a heterocyclic, a nucleoside or a polynucleotide.
  • the zwitterionic colipid contained in the liposomal mixture comprises a phosphatidylcholine (e.g., DMPC, DSPC, DOPC, DPPC, DAPC, POPC, OPPC, eggPC, soyPC, hydrogenated soyPC, and mixtures thereof).
  • the sterol contained in the liposomal mixture is selected from the group consisting of cholesterol, cholesterol derivatives, coprostanol, cholestanol, cholestane, cholesterolhemisuccinate, cholesterol sulfate, and mixtures thereof.
  • the cationic cardiolipin contained in the liposomal mixture is (R)-PLC-2.
  • the liposomal mixture contains 9-36 mole% of a cationic cardiolipin (e.g., about 54.0 mole % of DOPC), 36- 72 mole % of a zwitterionic lipid (e.g., about 16.0 mole % of (R)-PLC-2) and 9-36 mole % of a sterol (e.g., about 30 mole % of cholesterol).
  • a cationic cardiolipin e.g., about 54.0 mole % of DOPC
  • 36- 72 mole % of a zwitterionic lipid e.g., about 16.0 mole % of (R)-PLC-2
  • 9-36 mole % of a sterol e.g., about 30 mole % of cholesterol
  • the oligomer contained in the liposomal mixture comprises an oligomer that hybridizes to SEQ ID NO: 1249 or the complement thereof.
  • the oligomer can be SEQ ID NO:1250, 1251, 1252, 1253, 1267-1477 or the complement thereof.
  • the oligomer can be SEQ ID NO: 1250 or 1251 or the complement thereof.
  • a liposomal mixture that comprises an oligomer that hybridizes to SEQ ID NO:936 or the complement thereof, nucleotides 1-1000 of SEQ ID NO:936 or the complement thereof; SEQ ID NOs: 940, 943 or the complements thereof.
  • the liposomal mixture of this invention may further include a second oligomer, e.g., comprising one of SEQ ID NOs: 1250-1253 and 1270-1477 or the complements thereof, or selected from the group consisting of SEQ ID NOs: 2-281, 283-461, 463-935, 937-1080, and 1082-1277 and the complements thereof.
  • the oligonucleotides contained in the liposomal mixture are between 15 and 35 base pairs in length.
  • the mixture has a charge ratio from about 1.5 to about 3.0 (e.g., from about 2.0 to about 2.5).
  • the molar ratio of total lipids to oligomer in the mixture is from about 100 to about 600 (e.g., about 361) or from about 100 to about 180 (e.g., about 144).
  • the liposomal mixture further includes a stabilizer (e.g., tocopherol acid succinate) and/or an isotonicity adjuster (e.g., sucrose, trehalose, maltose, lactose, or glucose).
  • a stabilizer e.g., tocopherol acid succinate
  • an isotonicity adjuster e.g., sucrose, trehalose, maltose, lactose, or glucose.
  • the liposomal mixture has a pH value between about 5.1 and 8.0 (e.g., about 7.6) or the liposomes have a size between about 50 ⁇ m and about 500 ⁇ m (e.g., between about 80 and about 300 ⁇ m, or between about 90 and about 200 ⁇ m).
  • the liposomal mixture comprises the oligonucleotide PNT-100 (SEQ ID NO:1251) and the lipids DOPC, (R)-PLC-2, and cholesterol wherein the concentration of PNTlOO is about 2 mg/mL, the molar ratio of DOPC/(R)-PLC-2/cholesterol is about 54.0/16.0/30.0, and the total lipids, lipid to PNTlOO molar ratio and (+/-) charge ratio are about 30.0 mg/mL, 144 and 2.0, respectively.
  • the invention provides a method of introducing the oligonucleotide- liposome mixture to cells, tissue or a patient (including an animal or a human).
  • the mixture is administered to a cancer cell, a non-human animal or a human.
  • the mixture is introduced to an animal at a dosage of between 0.01 mg to 100 mg per kg of body weight.
  • the mixture is introduced to the animal one or more times per day or continuously.
  • the mixture is introduced to the animal via topical, pulmonary, or parenteral administration or via a medical device.
  • the mixture administered to the animal or cells further includes a known chemotherapy agent, and/or a cell targeting component.
  • Examples of known chemotherapy agents include Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, dacarbazine, Daunorubicin, Doxorubicin, Epirubicin, Etoposide, Idarubicin, Ifosfamide, Irinotecan, Lomustine, Mechlorethamine, Melphalan, Mitomycin C, Mitoxantrone, Oxaliplatin, Temozolamide and Topotecan.
  • Examples of cell targeting components include portions of peptides that mediate virus-cell fusion (e.g., DP 178) and small chemokines (e.g., SDF-I, RANTES).
  • the invention provides a method of preparing the liposomal delivery system.
  • the method comprises dispersing a cationic colipid, a zwitterionic colipid, cholesterol and a stabilizer in a solvent to form a dispersion, mixing an oligomer with an isotonicity adjuster to form a first mixture, combining the dispersion and the first mixture to form a second mixture, extruding the bulk liposomes comprising the solvent, the cationic colipid, the zwitterionic colipid, cholesterol, the stabilizer, the oligomer and the isotonicity adjuster from the second mixture and removing the solvent from the extruded liposomes.
  • the solvent is ethanol.
  • the invention provides another method of preparing the liposomal delivery system.
  • the method comprises dissolving a cationic cardiolipin analog, a zwitterionic lipid and a sterol in a solvent, adding an oligomer to the lipid mixture to form a dispersion, extruding the dispersion and removing a substantial portion of the solvent from the extruded liposomes.
  • the method further comprises adding a stabilizer to the lipid mixture.
  • the invention provides yet another method of preparing the liposomal delivery system.
  • the method comprises dissolving a cationic cardiolipin analog, a zwitterionic lipid and a sterol in a solvent to form a lipid mixture, mixing an isotonicity adjuster with an oligomer to form an oligomer mixture, combining the lipid mixture and the oligomer mixture to form a dispersion, extruding the dispersion and removing the solvent from the extruded liposomes.
  • the method further comprises adding a stabilizer to the lipid mixture.
  • the invention provides a liposomal mixture prepared by any of the methods described above.
  • Figure 1 shows inhibition of the growth of PC-3 xenografts by liposomal-PNT-100 (SEQ ID NO: 1251.
  • Figure 2 shows inhibition of the growth of WSU-DLCL2 cells in culture by liposomal PNT-100 (SEQ ID NO:1251 and PNT-104 (SEQ ID NO:1250).
  • Figure 3 shows inhibition of the growth of PC-3 cells in culture by liposomal PNT-100
  • Figure 4 shows Figure 3 shows percentage increase in tumor size in PC-3 xenografts following treatment with Liposomal SEQ ID NO: 1251 and TaxotereTM.
  • Figure 5 shows percentage increase in tumor size in PC-3 xenografts following treatment with SEQ ID NO: 1251 formulated with NeoPhectin-AT and TaxotereTM.
  • Figure 6A and 6B show NeoPhectin formulated CM-7 (SEQ ID NO:943) inhibition of the growth of MCF-7 breast cancer cells.
  • liposome refers to one or more lipids forming a complex, usually surrounded by an aqueous solution.
  • the liposomes may be uni, oligo or multi lamellar, containing an aqueous interior or a complex of lipids with a hydrophobic interior.
  • Liposomes may also include a drug, molecule or compound either bound to the liposomes or sequestered in the interior of the liposomes.
  • the molecules include, without limitation, oligonucleotides and/or other agents used to treat diseases such as cancer.
  • co-lipid refers to any hydrophobic material that may be combined with another lipid.
  • a "zwitterionic lipid” is a neutral lipid having electrical charges of opposite signs, delocalized or not on adjacent or nonadjacent atoms. Zwitterionic lipids have no uncharged canonical representations. Examples include phospholipids such as phosphatidyl choline and phosphatidyl ethanolamine.
  • a non-toxic anion is an atom or chemical group with a negative charge so that when it is part of a molecule, the anion does not confer toxicity on the molecule.
  • Examples include bromine, chlorine and iodine.
  • charge ratio is defined as all positive charges associated with the cationic lipid and all negative charges associated with the oligonucleotide backbone. The charge ratio does not take into consideration charges associated with the oligonucleotide base groups or with the zwitterionic cation.
  • sequestered, sequestering, or sequester refers to encapsulation, incorporation, or association of a drug, molecule, compound, including an oligonucleotide with the lipids of a liposome. It includes encapsulation within the liposome. It also includes part of the molecule in the interior of the liposome and part in the lipid portion of the liposome, or part sticking out of the liposomal exterior. It also includes molecules partially or totally embedded in the lipid portion of the liposome, and includes molecules associated with the liposomes, with all or part of the molecule associated with the exterior of the liposome.
  • aliphatic encompasses the terms alkyl, alkenyl, and alkynyl, each of which being optionally substituted as set forth below.
  • an "alkyl” group refers to a saturated aliphatic hydrocarbon group containing 1-8 (e.g., 1-6 or 1-4) carbon atoms. An alkyl group can be straight or branched.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, or 2-ethylhexyl.
  • An alkyl group can be substituted (i.e., optionally substituted) with one or more substituents such as halo, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, cycloaliphaticcarbonyl, (heterocycloaliphatic)carbonyl, nitro, cyano, amino, amido, acyl, sulfonyl, sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkoxy, or hydroxy.
  • substituents such as halo, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl,
  • substituted alkyls include carboxyalkyl (such as HOOC-alkyl, alkoxycarbonylalkyl and alkylcarbonyloxyalkyl), cyanoalkyl, hydroxyalkyl, alkoxyalkyl, acylalkyl, hydroxyalkyl, aralkyl, (alkoxyaryl)alkyl, (sulfonylamino)alkyl (such as (alkylsulfonylamino)alkyl), aminoalkyl, amidoalkyl, (cycloaliphatic)alkyl, cyanoalkyl, or haloalkyl.
  • carboxyalkyl such as HOOC-alkyl, alkoxycarbonylalkyl and alkylcarbonyloxyalkyl
  • cyanoalkyl such as HOOC-alkyl, alkoxycarbonylalkyl and alkylcarbonyloxyalkyl
  • cyanoalkyl such as HOOC-al
  • an "alkylene” group refers to an internal alkyl linking group having the structure -R- wherein R is an alkyl.
  • an "alkoxo” or “alkoxy” group refers to an alkyloxy group having the structure of -O-R wherein R is an alkyl group.
  • an "alkenyl” group refers to an aliphatic carbon group that contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and at least one double bond. Like an alkyl group, an alkenyl group can be straight or branched. Examples of an alkenyl group include, but are not limited to, allyl, isoprenyl, 2-butenyl, and 2-hexenyl.
  • An alkenyl group can be optionally substituted with one or more substituents such as halo, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, (cycloaliphatic)carbonyl, (heterocycloaliphatic)carbonyl, nitro, cyano, amino, amido, acyl, sulfonyl, sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy, heteroaryloxy, aralkyloxy, (heteroaryl)alkoxy, or hydroxy.
  • substituents such as halo, cycloaliphatic, heterocycloaliphatic, aryl, heteroary
  • an "alkenylene” group refers to an internal alkene linking group having the structure -R- wherein R is an alkene group.
  • an "alkynyl” group refers to an aliphatic carbon group that contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and has at least one triple bond.
  • An alkynyl group can be straight or branched. Examples of an alkynyl group include, but are not limited to, propargyl and butynyl.
  • An alkynyl group can be optionally substituted with one or more substituents such as halo, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, (cycloaliphatic)carbonyl, (heterocycloaliphatic)carbonyl, nitro, cyano, amino, amido, acyl, sulfonyl, sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy, heteroaryloxy, aralkyloxy, (heteroaryl)alkoxy, or hydroxy.
  • substituents such as halo, cycloaliphatic, heterocycloaliphatic, aryl, hetero
  • a "cycloalkyl” group refers to a saturated carbocyclic mono- or bicyclic (fused or bridged) ring of 3-10 (e.g., 5-10) carbon atoms.
  • Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, norbornyl, cubyl, octahydro-indenyl, decahydro-naphthyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2.]decyl, bicyclo[2.2.2]octyl, adamantyl, azacycloalkyl or ((aminocarbonyl)cycloalkyl)cycloalkyl.
  • a "cycloalkenyl” group refers to a non-aromatic carbocyclic ring of 3- 10 (e.g., 4-8) carbon atoms having one or more double bonds.
  • Examples of cycloalkenyl groups include cyclopentenyl, 1,4-cyclohexa-di-enyl, cycloheptenyl, cyclooctenyl, hexahydro- indenyl, octahydro-naphthyl, cyclohexenyl, cyclopentenyl, bicyclo[2.2.2]octenyl, and bicyclo[3.3.1 ]nonenyl.
  • a cycloalkyl or cycloalkenyl group can be optionally substituted with one or more substituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl], cycloaliphatic, (cycloaliphatic) aliphatic, heterocycloaliphatic, (heterocycloaliphatic) aliphatic, aryl, heteroaryl, alkoxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy, heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl, heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino, (cycloaliphatic)carbonylamino, ((cycloaliphatic)aliphatic)carbonylamino, (aryl)carbonylamino, (araliphatic)carbonylamino, (heterocycloalipha
  • cyclic moiety includes cycloaliphatic, heterocycloaliphatic, aryl, or heteroaryl, each of which has been defined previously.
  • a "halogen” or “halo” group refers to fluorine, chlorine, bromine, or iodine.
  • an “amino” group refers to -NR R wherein each of R and R is independently hydrogen, alkyl, cycloaliphatic, (cycloaliphatic)aliphatic, aryl, araliphatic, heterocycloaliphatic, (heterocycloaliphatic)aliphatic, heteroaryl, carboxy, sulfanyl, sulfinyl, sulfonyl, (aliphatic)carbonyl, (cycloaliphatic)carbonyl, ((cycloaliphatic)aliphatic)carbonyl, arylcarbonyl, (araliphatic)carbonyl, (heterocycloali ⁇ hatic)carbonyl,
  • amino groups include alkylamino, dialkylamino, and arylamino.
  • amino is not the terminal group (e.g., alkylcarbonylamino), it is represented by -NR X -.
  • R x has the same meaning as defined above.
  • saccharides refers to any naturally occurring or unnatural sugars like glucose, mannose, allose, ribose, fucose, arabinose, galactose, 2-deoxy sugars, 3- deoxy sugars, 4-deoxy sugars, disaccharide and polysaccharides.
  • oligosaccharide and “polysaccharide,” as known in the art, refer to saccharide polymers containing a small number (typically three to six) and higher number
  • oligosaccharide and polysaccharides include heparin of various molecualr weights and starch.
  • amino acid refers to any naturally occurring or unnatural amino acid. This definition is intended to embrace substituted ⁇ -amino acids as well as non- ⁇ -amino acids.
  • An ⁇ -amino acid is defined as an amino acid in which the amino group is attached to a carbon atom that is adjacent to the carboxylic acid group.
  • thio refers to the structure "-S-" when used as an internal linker, or the structure of "-SH” when used as a terminal group.
  • sulfinyl refers to -S(O)-R X when used terminally and -S(O)- when used internally, wherein R x is an aliphatic group.
  • each of the specific groups for the variables R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 1O , and other variables contained in formulae I, II, and III can be optionally substituted with one or more substituents described herein.
  • Each substituent of a specific group is further optionally substituted with one to three of halo, cyano, oxoalkoxy, hydroxyl, amino, nitro, aryl, haloalkyl, and alkyl.
  • an alkyl group can be substituted with alkylsulfanyl and the alkylsulfanyl can be optionally substituted with one to three of halo, cyano, oxoalkoxy, hydroxyl, amino, nitro, aryl, haloalkyl, and alkyl.
  • the cycloalkyl portion of (cycloalkyl)carbonylamino can be optionally substituted with one to three halo, cyano, alkoxy, hydroxyl, nitro, haloalkyl and alkyl.
  • substituted refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent.
  • substituents are described above in the definitions and below in the description of compounds and examples thereof.
  • an optionally substituted group can have a substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent can be either the same or different at every position.
  • a ring substituent such as a heterocycloalkyl
  • substituents envisioned by this invention are those combinations that result in the formation of stable or chemically feasible compounds.
  • an effective amount is defined as the amount required to confer a therapeutic effect on the treated patient, and is typically determined based on age, surface area, weight and condition of the patient.
  • the interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich et al., Cancer Chemother. Rep., 50: 219 (1966).
  • Body surface area can be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, New York, 537 (1970).
  • patient refers to a mammal, including a human.
  • structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
  • structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 1 C-enriched carbon are within the scope of this invention.
  • Such compounds are useful, for example, as analytical tools or probes in biological assays.
  • non-human animals refers to all non-human animals including, but are not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.
  • nucleic acid molecule refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA.
  • polynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides as used herein refers to, among others, single-and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions.
  • polynucleotide as used herein also encompasses triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • polynucleotide also includes DNAs or RNAs that contain one or more modified bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides,” “nucleic acid molecules” or “nucleic acid sequences” as those terms are intended herein.
  • the terms also encompass sequences that include any of the known base analogs of DNA and RNA.lt will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.
  • isolated nucleic acid sequence is meant a polynucleotide that is not immediately contiguous with either of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences.
  • the nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.
  • the term includes single and double stranded forms of DNA.
  • the term "gene” refers to a nucleic acid (e.g., DNA) sequence that includes coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA).
  • the polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragment are retained.
  • the term also encompasses the coding region of a structural gene and the sequences preceding and following the coding region, (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences.
  • the term "gene” encompasses both cDNA and genomic forms of a gene.
  • a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions” or “intervening sequences.”
  • Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers.
  • Introns are removed or "spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
  • mRNA messenger RNA
  • the mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
  • RNA expression refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through “translation” of mRNA.
  • Gene expression can be regulated at many stages in the process.
  • Up-regulation” or “activation” refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while “down- regulation” or “repression” refers to regulation that decrease production.
  • Molecules e.g., transcription factors
  • activators e.g., transcription factors
  • genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the niRNA transcript).
  • the 5 1 flanking region (or upstream region) may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene.
  • the 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation.
  • nucleic acid molecule encoding refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
  • oligonucleotide as used herein is defined as a molecule included of two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size of an oligonucleotide will depend on many factors, including the ultimate function or use of the oligonucleotide. Oligonucleotides can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth.
  • oligonucleotides are one form of the compounds, the present invention comprehends other oligomeric oligonucleotide compounds, including but not limited to oligonucleotide mimetics such as are described below.
  • the oligonucleotide compounds in accordance with this invention preferably comprise from about 15 to about 35 nucleobases (i.e., from about 15 to about 35 linked bases), although both longer and shorter sequences may find use with the present invention.
  • Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base”
  • “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases.
  • Another modification of the oligonucleotides of the present invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, (e.g., hexyl-S-tritylthiol), a thiocholesterol, an aliphatic chain (e.g., dodecandiol or undecyl residues), a phospholipid, (e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or a polyethylene glycol chain or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl- oxycholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a thioether, (e
  • oligonucleotides containing the above-described modifications are not limited to the oligonucleotides described above. Any suitable modification or substitution may be utilized. [077] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.
  • the present invention also includes pharmaceutical compositions and formulations that include the antisense compounds of the present invention as described below.
  • oligonucleotides are "antigenes.”
  • the term “antigene” refers to an oligonucleotide that hybridizes to the promoter region of a gene. In some embodiments, the hybridization of the antigene to the promoter inhibits expression of the gene.
  • promoter is meant a sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5 ' or 3' regions of the gene. Both constitutive and inducible promoters, are included in the invention (see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987).
  • inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
  • promoters derived from the genome of mammalian cells e.g., metallothionein promoter
  • mammalian viruses e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention.
  • the "regulatory region" of a gene is any part of a gene that regulates the expression of a gene, including, without limitation, transcriptional and translational regulation.
  • the regions include without limitation the 5' and 3' regions of genes, binding sites for regulatory factors, including without limitation transcription factor binding sites.
  • the regions also include regions that are as long as 20,000 or more base pairs upstream or downstream of the translational start site, so long as the region is involved in any way in the regulation of the expression of the gene.
  • the region may be as short as 20 base pairs or as long as thousands of base pairs.
  • transformation or transfection is meant a permanent or transient genetic change induced in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell).
  • new DNA i.e., DNA exogenous to the cell.
  • a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.
  • transformed cell or "host cell” is meant a cell (e.g., prokaryotic or eukaryotic) into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding a polypeptide of the invention (i.e., a Methuselah polypeptide), or fragment thereof.
  • a cell e.g., prokaryotic or eukaryotic
  • a DNA molecule encoding a polypeptide of the invention i.e., a Methuselah polypeptide
  • the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence “A-G-T,” is complementary to the sequence “T-C-A.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
  • the term “completely complementary,” for example, when used in reference to an oligonucleotide of the present invention refers to an oligonucleotide where all of the nucleotides are complementary to a target sequence (e.g., a gene).
  • a target sequence e.g., a gene
  • the term “partially complementary,” refers to a sequence where at least one nucleotide is not complementary to the target sequence. Preferred partially complementary sequences are those that can still hybridize to the target sequence under physiological conditions.
  • the term “partially complementary” refers to sequences that have regions of one or more non-complementary nucleotides both internal to the sequence or at either end.
  • Sequences with mismatches at the ends may still hybridize to the target sequence.
  • the term "homology" refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity).
  • a partially complementary sequence is a nucleic acid molecule that at least partially inhibits a completely complementary nucleic acid molecule from hybridizing to a target nucleic acid is "substantially homologous.” The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
  • a substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous nucleic acid molecule to a target under conditions of low stringency.
  • a substantially complementary sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely complementary nucleic acid molecule to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target that is substantially non- complementary (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
  • substantially homologous refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
  • substantially homologous refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
  • hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T m of the formed hybrid, and the G:C ratio within the nucleic acids.
  • T m is used in reference to the "melting temperature.”
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • the equation for calculating the T m of nucleic acids is well known in the art.
  • T 1n 81.5 + 0.41(% G + C)
  • Other references include more sophisticated computations that take structural as well as sequence characteristics into account for the calculation of T m .
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
  • a substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous nucleic acid molecule to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target that is substantially non-complementary (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non- complementary target.
  • substantially homologous refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
  • stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted.
  • low stringency conditions a nucleic acid sequence of interest will hybridize to its exact complement, sequences with single base mismatches, closely related sequences (e.g., sequences with 90% or greater homology), and sequences having only partial homology (e.g., sequences with 50-90% homology).
  • intermediate stringency conditions a nucleic acid sequence of interest will hybridize only to its exact complement, sequences with single base mismatches, and closely related sequences (e.g., 90% or greater homology).
  • a nucleic acid sequence of interest will hybridize only to its exact complement, and (depending on conditions such a temperature) sequences with single base mismatches. In other words, under conditions of high stringency the temperature can be raised so as to exclude hybridization to sequences with single base mismatches.
  • physiological conditions refers to specific stringency conditions that approximate or are conditions inside an animal (e.g., a human).
  • exemplary physiological conditions for use in vitro include, but are not limited to, 37°C, 95% air, 5% CO 2 , commercial medium for culture of mammalian cells (e.g., DMEM media available from Gibco, MD), 5-10% serum (e.g., calf serum or horse serum), additional buffers, and optionally hormone (e.g., insulin and epidermal growth factor).
  • isolated means altered “by the hand of man” from its natural state; i.e., if it occurs in nature, it has been changed or removed from its original environment or both.
  • an isolated nucleotide or “isolated polynucleotide” refers to a nucleic acid sequence that is identified and separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Isolated nucleic acid is such present in a form or setting that is different from that in which it is found in nature.
  • non-isolated nucleic acids are nucleic acids such as DNA and RNA found in the state they exist in nature.
  • a given DNA sequence e.g., a gene
  • RNA sequences such as a specific mRNA sequence encoding a specific protein
  • isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the given protein where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.
  • the isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form.
  • the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).
  • the term “Western blot” refers to the analysis of protein(s) (or polypeptides) immobilized onto a support such as nitrocellulose or a membrane.
  • the proteins are run on acrylamide gels to separate the proteins, followed by transfer of the protein from the gel to a solid support, such as nitrocellulose or a nylon membrane.
  • the immobilized proteins are then exposed to antibodies with reactivity against an antigen of interest.
  • the binding of the antibodies may be detected by various methods, including the use of radiolabeled antibodies.
  • the term “cell culture” refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.
  • in vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment.
  • in vitro environments can consist of, but are not limited to, test tubes and cell culture.
  • in vivo refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.
  • the term "under conditions such that expression of a gene is inhibited” refers to conditions where an oligonucleotide of the present invention hybridizes to a gene (e.g., the promoter region of the gene) and inhibits transcription of the gene by at least 10%, at least 25%, at least 50% or at least 90% relative to the level of transcription in the absence of the oligonucleotide.
  • the present invention is not limited to the inhibition of expression of a particular gene.
  • Exemplary genes include, but are not limited to, c-ki-ras, c-Ha-ras, c-myc, her-2, TGF- ⁇ , and bcl-2.
  • the term "under conditions such that growth of a cell is reduced” refers to conditions where an oligonucleotide of the present invention, when administered to a cell (e.g., a cancer) reduces the rate of growth of the cell by at least 10%, at least 25%, at least 50% or at least 90% relative to the rate of growth of the cell in the absence of the oligonucleotide.
  • test compound and “candidate compound” refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g., cancer). Test compounds include both known and potential therapeutic compounds.
  • test compound can be determined to be therapeutic by screening using the screening methods of the present invention.
  • test compounds include antisense compounds.
  • known chemotherapeutic agents refers to compounds known to be useful in the treatment of disease (e.g., cancer).
  • chemotherapeutic agents affective against cancer include, but are not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, taxotere, vincristine, vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol (DES).
  • daunorubicin dactinomycin
  • doxorubicin bleomycin
  • mitomycin nitrogen mustard
  • chlorambucil chlorambucil
  • melphalan cyclophosphamide
  • 6-mercaptopurine 6-thioguanine
  • cytarabine CA
  • (R)-PLC-2 1 ,3-Bis-(l ,2-bis-tetradecyloxy-propyl-3- dimethylethoxyammonium bromide)-propan-2-ol.
  • DMPC Dimyristoylphosphatidylcholine [0106] DPPC Dipalmitoylphosphatidylcholine [0107] DSPC Distearoylphosphatidylcholine [0108] POPC Palmitoyl-oleoylphosphatidylcholine [0109] OPPC l-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine [0110] DOPC Dioleoylphosphatidylcholine [0111] DAPC Diarachidonoylphosphatidylcholine [0112] DOPE Dioleoylphosphatidylethanolamine [0113] DMPE Dimyristoylphosphatidylethanolamine [0114] DPPE Dipalmitoylphosphatidylethanolamine [0115] CHEMS Cholesterolhemisuccinate [0116] DC-Choi 3- ⁇ -[N-(N',N'-dimethyle
  • CTAB Cetyl-trimethylammoniumbromide
  • CPyC Cetyl-pyridiniumchloride [0125] DOTAP (1 ,2-dioleoyloxypropyl)-N,N,N-trimethylammonium salt [0126] DMTAP ( 1 ,2-dimyristoyloxypropyl)-N,N,N-trimethylammonium salt [0127] DPTAP (1 ,2-dipalmitoyloxypropyl)-N,N,N-trimethylammonium salt [0128] DOTMA (1 ,2-dioleyloxypropyl)-N,N,N-trimethylammonium chloride) [0129] DORIE (1 ,2-dioleyloxypropyl)-3 dimethylhydroxyethyl ammoniumbromide) [0130] DDAB Dimethyldioctadecylammonium bromide
  • DPIM 4-(2,3 -bis-palmitoyloxy-propyl)- 1 -methyl- 1 H-imidazole [0132] CHIM Histaminyl-Cholesterolcarbamate [0133] HCChol N ⁇ -Histidinyl-Cholesterolcarbamate [0134] HistChol N ⁇ -Histidinyl-Cholesterol-hemisuccinate [0135] AC Acylcarnosine, Stearyl- & Palmitoylcarnosine [0136] HistDG 1,2-Dipalmitoylglycerol-hemisuccinat-N-Histidinyl- hemisuccinate, & Distearoyl-, Dimyristoyl-, Dioleoyl-, or palmitoyl-oleoylderivatives
  • EDTA-Chol cholesterol ester of ethyl enediaminetetraacetic acid [0140] Hist-PS N ⁇ -histidinyl-phosphatidylserine [0141] TC-Chol 3- ⁇ -[N-(N',N'-trimethylaminoethane)carbamoyl] cholesterol [0142] DOSPER (1.3-dioleoyloxy-2-(6-carboxy-spermyl)-propylarnide [0143] DOSC (l,2-dioleoyl-3-succinyl-sn-glyceryl choline ester) [0144] DOGSDO (1 ,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide ornithine)
  • DMGSucc l,2-Dimyristoylglycerol-3-hemisuccinate [0148] DPGSucc 1 ,2-Dipalmitoylglycerol-3 -hemisuccinate
  • the present invention provides antigene inhibitors of oncogenes.
  • the present invention is not limited to the inhibition of a particular oncogene. Indeed, the present invention encompasses antigene inhibitors to any number of oncogenes including, but not limited to, those disclosed herein.
  • c-Ha-ras One gene which has captured the attention of many scientists is the human proto- oncogene, c-Ha-ras. This gene acts as a central dispatcher, relaying chemical signals into cells and controlling cell division. Ras gene alteration may cause the gene to stay in the "on" position.
  • the ras oncogene is believed to underlie up to 30% of cancer, including colon cancer, lung cancer, bladder and mammary carcinoma (Bos, Cancer Res. 49:4682-4689 [1989]). The ras oncogene has therefore become a target for therapeutic drugs.
  • Oligonucleotides complementary to the 5' flanking region of the c-Ha-ras RNA transcript have shown to inhibit tumor growth in nude mice for up to 14 days (Gray et al, Cancer Res. 53:577-580 [1993]). It was recently reported that an antisense oligonucleotide directed to a point mutation (G>C) in codon 12 of the c-Ha-ras mRNA inhibited cell proliferation as well as tumor growth in nude mice when it was injected subcutaneously (U.S. Pat. No. 5,576,208; U.S. Pat. No. 5,582,986; Schwab et al, Proc. Natl. Acad. Sci.
  • the -her-2 also known as neu oncogene or erbB-2
  • oncogene encodes a receptor-like tyrosine kinase (RTK) that has been extensively investigated because of its role in several human carcinomas (Hynes and Stern, Biochim. et Biophy. Acta 1198:165-184 [1994]; Dougall et al, Oncogene 9:2109-2123 [1994]) and in mammalian development (Lee et al, Nature 378:394-398 [1995]).
  • RTK receptor-like tyrosine kinase
  • the sequence of the HER-2 protein was determined from a cDNA that was cloned by homology to the epidermal growth factor receptor (EGFR) mRNA from placenta (Coussens et al, Science 230:1132-1139 [1985]) and from a gastric carcinoma cell line (Yamamoto et al, Nature 319:230-234 [1986]).
  • EGFR epidermal growth factor receptor
  • the her-2 mRNA was shown to be about 4.5 kb (Coussens et al, Science 230:1132-1139 [1985]; Yamamoto et al, Nature 319:230-234 [1986]) and encodes a transmembrane glycoprotein of 185 kDa in normal and malignant human tissues (pl85HER-2) (Hynes and Steen, Biochim. et Biophys. Acta 1198:165-184 [1994]; Dougall et al, Oncogene 9:2109-2123 [1994]).
  • HER-2 causes phenotypic transformation of cultured cells (DiFiore et al, Science 237:178-182 [1987]; Hudziak et al, Proc. Natl. Acad. Sci. USA 84:7159-7163 [1987]) and has been associated with aggressive clinical progression of breast and ovarian cancer (Slamon et al, Science 235:177- 182 [1987]; Slamon et al, Science 244:707-712 [1989]).
  • Her-2 is one of the most frequently altered genes in cancer.
  • transmembrane receptor also known as pi 85
  • tyrosine kinase activity is a member of the epidermal growth factor (EGF) family, and thus is related to the epidermal growth factor receptor (EGFR or HER-I).
  • EGF epidermal growth factor
  • HER-2 Aberrant her-2 gene expression is present in a wide variety of cancers and are most common in breast, ovarian and gastric cancers.
  • HER-2 is overexpressed in 25-30% of all human breast and ovarian cancers. Levels of HER-2 overexpression correlate well with clinical stage of breast cancer, prognosis and metastatic potential. Overexpression of HER-2 is associated with lower survival rates, increased relapse rates and increased metastatic potential. Tan et al, (Cancer Res., 57:1199 [1997]) have shown that overexpression of the HER-2 gene increases the metastatic potential of breast cancer cells without increasing their transformation ability.
  • HER-2 Aberrant expression of HER-2 includes both increased expression of normal HER-2 and expression of mutant HER-2.
  • Activation of the her-2 proto-oncogene can occur by any of three mechanisms—point mutation, gene amplification and overexpression. Gene amplification is the most common mechanism. Unlike the other EGF family members for whom ligand activation is necessary for promoting transformation, overexpression of HER-2 alone is sufficient for transformation (Cohen, et al, J. Biol. Chem., 271:30897 [1996]).
  • Several therapeutic approaches have been used to reduce levels of the her-2 gene product.
  • the adenovirus type 5 gene product ElA has been studied as a potential therapeutic using a breast cancer model in nude mice.
  • This gene product can repress her-2/neu overexpression by repressing her-2/neu promoter activity, and suppress the tumorigenic potential of /zer-2/Weu-overexpressing ovarian cancer cells.
  • ElA delivered either by adenovirus or liposome significantly inhibited tumor growth and prolonged mouse survival compared with the controls (Chang et al, Oncogene 14:561 [1997]).
  • Oligonucleotides have also been used to study the function of HER-2.
  • a triplex- forming oligonucleotide targeted to the her-2 promoter, 42 to 69 nucleotides upstream of the mRNA transcription start site was found to inhibit HER-2 expression in vitro (Ebbinghaus et al, J. Clin. Invest., 92:2433 [1993]).
  • Porumb et al (Cancer Res., 56:515 [1996]) also used a triplex-forming oligonucleotide targeted to the same her-2 promoter region. Decreases in her- 2 mRNA and protein levels were seen in cultured cells. Juhl et al. (J. Biol.
  • Chem., 272:29482 [1997]) used ⁇ a ⁇ i-her-2 ribozymes targeted to a central region of the her-2 RNA just downstream of the transmembrane region of the protein to demonstrate a reduction in her-2 mRNA and protein levels in human ovarian cancer cells. A reduction in tumor growth in nude mice was also seen.
  • Colomer et al. (Br. J. Cancer, 70:819 [1994]) showed that phosphodiester antisense oligonucleotides targeted at or immediately downstream of, the translation initiation codon inhibited proliferation of human breast cancer cells by up to 60%.
  • Wiechen et al (Int. J. Cancer 63:604 [1995]) demonstrated that an 18-nucleotide phosphorothioate oligonucleotide targeted to the coding region, 33 nucleotides downstream of the translation initiation codon, of her-2 reduced anchorage- independent growth of ovarian cancer cells.
  • Bertram et al Biochem. Biophys. Res.
  • the c-myc gene product is encoded by an immediate early response gene, the expression of which can be induced by various mitogens. C-myc expression is involved in the signal transduction pathways leading to cell division. Studies have demonstrated that proliferating cells have higher levels of c-myc mRNA and c-myc protein than do quiescent cells. Antibodies directed against the human c-myc protein are known to inhibit DNA synthesis in nuclei isolated from human cells. Conversely, constitutive expression of c-myc produced by gene transfer inhibits induced differentiation of several cell lines. Constitutive expression of c-myc predisposes transgenic mice to the development of tumors.
  • c-myc gene product may play a proliferative role in SMCs.
  • Balloon de-endothelialization and injury of rat aortas is known to increase c-myc mRNA expression of vascular SMC prior to their subsequent proliferation and migration.
  • SMCs in culture proliferate when exposed to several mitogens, including PDGF, FGF, EGF, IGF-I and to serum. Each of these mitogens has been found to be capable of increasing the expression in other cell lines of either c-myc protein, c-myc mRNA, or both.
  • blood serum has been found to increase c-myc mRNA levels in SMCs.
  • Harel-Bellan et al (J. Immun. 140; 2431-2435 (1988)) demonstrated that antisense oligonucleotides complementary to c-myc mRNA effectively inhibited the translation thereof in human T cells. These T cells were prevented from entering the S phase of cell division, c- myc proto-oncogene sequences are described in Marcu et al, Ann. Rev. Biochem., 61:809-860 [1992]; Watt et al, Nature, 303:725-728 [1983)]; Battey et al, Cell, 34:779-787 (1983); and Epstein et al, NTIS publication PB93- 100576
  • the bcl-2 gene is overexpressed, and may be associated with tumorigenicity (Tsujimoto et al, Science 228:1440-1443 [1985]). High levels of expression of the bcl-2 gene have been found in all lymphomas with t (14; 18) chromosomal translocations including most follicular B cell lymphomas and many large cell non-Hodgkin's lymphomas.
  • TGF- ⁇ Transforming Growth Factor Alpha
  • EGF- ⁇ is a polypeptide of 50 amino acids. It was first isolated from a retrovirus-transformed mouse cell line and subsequently was identified in human tumor cells, in early rat embryo cells and in cell cultures from the human pituitary gland. TGF- ⁇ is closely related to Epidermal Growth Factor (EGF), both structurally and functionally, and both bind to the same receptor, i.e., Epidermal Growth Factor Receptor (EGFR).
  • EGF Epidermal Growth Factor
  • TGF- ⁇ is a 50 amino acid polypeptide having about 40% homology of residues with EGF. Both peptides are characterized by three well defined loops (denoted A, B and C) and have three intramolecular disulphide bonds.
  • EGF Receptor Epidermal Growth Factor Receptor
  • the EGF Receptor is a Type 1 receptor tyrosine kinase.
  • the EGF Receptor and its ligands are of interest for their roles in normal physiological processes as well as in hyperproliferative and neoplastic diseases.
  • TGF- ⁇ The in vivo precursor of TGF- ⁇ is a 160 amino acid residue membrane-bound protein (pro-TGF-. alpha.) that is cleaved to yield a soluble compound (Massague, J. Biol. Chem., 265:21393-21396 [1990]). This cleavage removes an extracellular portion comprised of 50 amino acids with a molecular weight of 6 Kd and is considered to be an important regulatory event (Pandiella et al, Proc. Natl. Acad. Sci. USA, 88:1726-1730 [1990]) that can be stimulated by phorbol esters acting via protein kinase C (Pandiella et al, J. Biol.
  • TGF- ⁇ Cultured human prostatic tumor lines contain elevated levels of TGF- ⁇ mRNA and proliferate in response to TGF- ⁇ (Wilding et al, The Prostate, 15:1-12 [1989]). TGF- ⁇ appears to have both autocrine and paracrine function, stimulating physiologic activities such as cell division and angiogenesis. When induced in transgenic mice, TGF- ⁇ produced epithelial hyperplasia and focal dysplastic changes that resembled carcinoma in situ (Sandgren et al, Cell, 61:1121-1135 [1990]).
  • KRAS c-Ki-Ras
  • the transforming gene product displayed an electrophoretic mobility in SDS- polyacrylamide gels that differed from the mobility of KRAS transforming proteins in other tumors. Thus, a previously undescribed mutation was responsible for activation of KRAS in this ovarian carcinoma.
  • Rodenhuis et al. (New Eng. J. Med. 317: 929 (1987)) used an assay based on oligonucleotide hybridization following an in vitro amplification step. Genomic DNA was examined from 39 tumor specimens obtained at thoracotomy. The KRAS gene was found to be activated by point mutations in codon 12 in 5 of 10 adenocarcinomas.
  • the present invention is not limited to the oncogenes described above.
  • the methods of the present invention are suitable for use with any oncogene with a known promoter region.
  • the present invention is not limited to the targeting of oncogenes.
  • the methods and compositions of the present invention find use in the targeting of any gene that it is desirable to down regulate the expression of.
  • the genes to be targeted include, but are not limited to, an immunoglobulin or antibody gene, a clotting factor gene, a protease, a pituitary hormone, a protease inhibitor, a growth factor, a somatomedian, a gonadotrophin, a chemotactin, a chemokine, a plasma protein, a plasma protease inhibitor, an interleukin, an interferon, a cytokine, a transcription factor, or a pathogen target (e.g., a viral gene, a bacterial gene, a microbial gene, a fungal gene).
  • a pathogen target e.g., a viral gene, a bacterial gene, a microbial gene, a fungal gene.
  • genes include, but are not limited to, ADAMTS4, ADAMTS 5, APOAl, APOE, APP, B2M, COX2, CRP, DDX25, DMCl, FKBP8, GHl, GHR, IAPP, IFNAl, IFNG, ILl, 1110, IL12, IL13, IL2, IL4, IL7, IL8, IPW, MAPK14, Meil, MMP13, MYD88, NDN, PACE4, PRNP, PSENl, PSEN2, RAD51, RAD51C, SAP, SNRPN, TLR4, TLR9, TTR, UBE3A, VLA-4, and PTP-IB, c-RAF, m-TOR, LDL, VLDL, ApoB-100, HDL, VEGF, rhPDGF-BB, NADs, ICAM-I, MUCl, 2-dG, CTL, PSGL-I, E2F, NF-k
  • pathogens include, but are not limited to, Human Immunodeficiency virus, Hepatitis B virus, hepatitis C virus, hepatitis A virus, respiratory syncytial virus, pathogens involved in severe acute respiratory syndrome, west nile virus, and food borne pathogens (e.g., E. col ⁇ ).
  • Cationic liposomes are a class of liposomes that have a cationic charge. They are thought to interact electrostatically with negatively charged nucleic acid sequences to form complexes that facilitate penetration of these agents into cells.
  • some cationic liposomal delivery systems are unstable in serum and have fairly high toxicity. Liposomes containing cardiolipin and cardiolipin analogues have been shown to be more stable in blood and have lower toxicity than some other liposomes containing cationic lipids.
  • Cationic liposomes of the present invention include (1) cationic lipids (e.g., a single lipid or a mixture of cationic lipids, (2) zwitterionic lipids, (3) sterols, and (4) one or more oligonucleotides, and, optionally, (5) a stabilizer and (6) an isotonicity adjuster.
  • a cationic lipid can be any lipid, including a substituent, such as one or more functional groups, that exhibits a cationic charge.
  • cationic substituents include, without limitation, amines (e.g., primary, secondary, tertiary, quaternary and cyclic).
  • the cationic lipids can be cardiolipin or cardiolipin analogs and/or other cationic lipids.
  • the cardiolipin analogs can be optically pure or their diasteroisomers. The analogs have the general formula I
  • n can be 1 or 2;
  • Z 1 , Z 2 , Z 3 , and Z 4 can be independently -O-C(O)-, -O-, -S- , or -NH-C(O)-;
  • R 1 , R 2 , R 3 , and R 4 are independently H, C 1 to C 32 saturated or unsaturated aliphatic, optionally substituted with one or more hydroxy, amino, sulfmyl, alkoxy, cycloaliphatic, PEG, halo, or combinations thereof;
  • R 5 is H, aliphatic, alkoxy, cycloaliphatic, alkanoyl, alkenoyl, or alkynoyl, each optionally substituted with one or more hydroxy, amino (e.g., dimethyamino), thio (e.g., -SH), sulfinyl (e.g., -S(O)-CH 3 or -S(O)-), al
  • R 6 and R 7 can be the same or different and are independently absent or comprise a linker comprising a C 1 to C 32 alkyl, cycloalkyl, or alkyloxy group, each optionally substituted with one or more hydroxy, amino, thio, epoxy, cycloalkyl, PEG, halo, or combinations thereof.
  • the Rg groups can be the same or different and can independently include a C 1 to C 25 saturated or unsaturated aliphatic or alkoxy, each optionally substituted with one or more hydroxy, amino, sulfinyl, alkoxy, cycloaliphatic, PEG, halo, or combinations thereof.
  • X is a non-toxic anion such as chloride, bromide, iodide and the like.
  • R 9 and R 10 can be the same or different and are independently absent or comprise a R 9 and R 1O are independently absent or comprise a linker comprising a Ci to C 32 aliphatic, cycloaliphatic, or alkoxy, each optionally substituted with one or more hydroxy, amino, sulfinyl, alkoxy, cycloaliphatic, PEG, halo, or combinations thereof.
  • B can be a non-toxic cation or a structure having the general formula IV
  • R 11 is a C 2 to C 32 alkylene, alkenylene, or alkoxo, each optionally substituted with one or more hydroxy, amino, sulfinyl, alkoxy, cycloaliphatic, PEG, halo, or combinations thereof.
  • R 12 , Ri 3 and Rj 4 are independently H, aliphatic, alkoxy, cycloaliphatic, alkanoyl, alkenoyl, or alkynoyl, each optionally substituted with one or more hydroxy, amino, sulfinyl, alkoxy, cycloaliphatic, PEG, halo, or combinations thereof; an amino acid, a peptide, a peptidoniimetic moiety, a dipeptide, a polypeptide, a protein, an oligosaccharide or polysaccharide, a polyamine, a heterocyclic, a nucleoside or a polynucleotide.
  • Ri, R 2 , R 3 , and R 4 are the same or different, and can be independently H, C 1 to C 32 saturated or unsaturated aliphatic (e.g., propyl or hexenyl), optionally substituted with one or more hydroxy, amino (e.g., diethylamino), sulfinyl (e.g., - S(O)-Et), alkoxy (e.g., ethoxy), cycloaliphatic (e.g., cyclohexyl), PEG (e.g., -(CH 2 CH 2 O) 20 -), halo (e.g., chloro), or combinations thereof.
  • C 1 to C 32 saturated or unsaturated aliphatic e.g., propyl or hexenyl
  • amino e.g., diethylamino
  • sulfinyl e.g., - S(O)-Et
  • alkoxy e.
  • R 6 and R 7 independently are absent or comprise a linker comprising a C 1 to C 32 alkyl (e.g., -(CH 2 ) 4 -), cycloalkyl, or alkyloxy group, each optionally substituted with one or more hydroxy, amino, thio, epoxy, cycloalkyl, PEG, halo, or combinations thereof.
  • a linker comprising a C 1 to C 32 alkyl (e.g., -(CH 2 ) 4 -), cycloalkyl, or alkyloxy group, each optionally substituted with one or more hydroxy, amino, thio, epoxy, cycloalkyl, PEG, halo, or combinations thereof.
  • R 5 is H, aliphatic, alkoxy, cycloaliphatic, alkanoyl, alkenoyl, or alkynoyl, each optionally substituted with one or more hydroxy, amino, sulfinyl, epoxy, cycloalkyl, PEG, halo, or combinations thereof; an amino acid, a peptide, a peptidomimetic moiety, a dipeptide, a polypeptide, a protein, an oligosaccharide or polysaccharide, a polyamine, a heterocyclic, a nucleoside or a polynucleotide.
  • At least one of R 5 , R 6 , R 7 and R 8 comprises an optionally substituted alkyl or optionally substituted alkyloxy group.
  • at least one of R 6 or R 7 (or both) can comprise an optionally substituted polyalkyloxy group containing from 1 to 500 alkyloxy groups, such as from 1 to 100 alkyloxy groups.
  • at least one of R 5 and R 8 comprises an optionally substituted alkyl and at least one ofR 6 and R 7 comprises an optionally substituted polyalkyloxy group.
  • at least one of R 6 and R 7 (or both) is ethoxy (mono PEG).
  • an R 5 substituent is an amino acid, folic acid, saccharide, peptide, polysaccharide, polypeptide, protein, polyamine or peptidomimetic moiety. Specific examples of such an R 5 substituent include histone, spermine, spermidine or a derivative thereof.
  • an R 5 substitutent is a saccharide attached as an O- glycoside or C-glycoside, such as glucose, mannose, galactose, ribose, arabinose, allose, fucose, a 2-deoxy sugar or the like.
  • an R 5 substituent can include an L-or D-alpha amino acid having a positively charged group on the side chain, such as, for example, arginine, histidine, lysine, ornithine or analogs thereof.
  • an R5 substituent comprises an amino acid, saccharide, peptide, polysaccharide, polypeptide, protein, polyamine or peptidomimetic moiety having one or more positive charge.
  • the two R 5 groups can be the same or different and can independently comprise such groups as set forth above.
  • the Rg groups are CH 3 .
  • At least one OfR 5 , R 6 , R 7 and R 8 comprises a substituted alkyl or substituted alkyloxy group.
  • Z 1 and Z 2 can be - 0-C(O)- or -O-.
  • R 1 , R 2 , R 3 and R 4 are the same and are a C 1 to C 32 saturated and/or unsaturated alkyl group, e.g., an alkyl group of 10 to 24 carbon atoms.
  • "X" can be a chloride or bromide ion.
  • n 1, R 6 and R 7 are absent, R 5 is H, R 8 is CH 3 , X is bromide, Z 1 and Z 2 are oxygen.
  • R 1 , R 2 , R 3 , and R 4 can be the same or different and can be independently H or C 1 to C 32 alkyl (e.g., methyl), alkenyl (e.g., ethylenyl), or alkynyl.
  • strcuture VI Shown below in strcuture VI is another exemplary compound (a cationic cardiolipin ester) according to general formula I, wherein n is 1, R 6 and R 7 are absent, R 5 is H, R 8 is CH 3 , X is bromide, and Z 1 to Z 4 are -O-C(O)-.
  • R 1 , R 2 , R 3 , and R 4 can be the same or different and can be independently H or C 1 to C 32 alkyl, alkenyl, or alkynyl groups.
  • n 1
  • R 6 is absent
  • R 7 is OCH 2 CH 2
  • R 5 is H
  • R 8 is CH 3
  • X is bromide
  • Z 1 to Z 4 are oxygen.
  • Rj, R 2 , R 3 , and R 4 can be the same or different and can be independently H or C 1 to C 32 aliphatic (e.g., methyl, ethylenyl, or butynyl).
  • all OfR 1 , R 2 , R 3 , and R 4 can be C 14 H 2 C 1 , resulting in a cationic cardiolipin ether analog (which is (R)-PLC-2) as shown in structure XVIII.
  • n 1, R 5 is H, R 6 is absent, R 7 is -OCH 2 CH 2 , R 8 is CH 3 , X is bromide, Z L to Z 4 are-O-C(O)-.
  • R 1 , R 2 , R 3 , and R 4 can be the same or different and they can be independently H or Ci to C 31 aliphatic (e.g., methyl, ethylenyl, or butynyl).
  • n 1
  • R 6 is OCH 2 CH 2
  • R 7 is absent
  • R5 is H
  • R 8 is CH 3
  • X is bromide
  • Z 1 to Z 4 are oxygen.
  • R 1 , R 2 , R 3 , and R 4 can be the same or different and can be independently H or C 1 to C 32 aliphatic (e.g., methyl, ethylenyl, or butynyl).
  • X is another exemplary compound (a cationic cardiolipin ester analog) according to formula I, wherein n is 1, R 6 is OCH 2 CH 2 , and R 7 is absent, R 5 is H, R 8 is CH 3 , X is bromide, Zi to Z 4 are -O-C(O)-.
  • R 1 , R 2 , R 3 , and R 4 can be the same or different and can be indepdently H or C 1 to C 3 i aliphatic (e.g., methyl, ethylenyl, or butynyl).
  • R 1 , R 2 , R 3 , and R 4 can be the same or different and can be independently H or C 1 to C 3 2 aliphatic.
  • XII is another exemplary compound (a cationic cardiolipin ester) according to formula I where n is 1, both R 6 and R 7 are OCH 2 CH 2 , R 5 is H, R 8 is CH 3 , X is bromide, Z 1 to Z 4 are -O-C(O)-.
  • R 1 , R 2 , R 3 , and R 4 can be the same or different and can be independently H or C 1 to C 31 aliphatic.
  • XIII is another exemplary compound (a cationic cardiolipin variant ether) according to formula I, wherein n is 2, both R 6 and R 7 are absent, R 5 is H, R 8 is CH 3 , X is bromide, Z 1 to Z 4 are oxygen.
  • R 1 , R 2 , R 3 , and R 4 can be the same or different and can be independently H or C 1 to C 32 aliphatic.
  • XIV is another exemplary compound (a cationic cardiolipin variant ester) according to formula I wherein n is 2, both R 6 and R 7 are absent, R 5 is H, R 8 is CH 3 , X is bromide, Z 1 to Z 4 are -O-C(O)-.
  • R 1 , R 2 , R 3 , and R 4 can be the same or different and can be independently H or C 1 to C 3 i aliphatic.
  • the invention provides cationic cardiolipin having structure
  • R 1 , R 2 , R 3 , R 4 , R 9 , R 10 , X, Z 1 , Z 2 , Z 3 , and Z 4 can be as described with reference to Formula I.
  • R 5 , R 12 , R 13 , and R 14 can be as described above with reference to R 5 in structure I.
  • Ri 1 can be, e.g., C 2 -C 32 , alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkyloxy or substituted alkyloxy.
  • XVI Shown below in structure XVI is an exemplary compound (a cationic cardiolipin variant ether) according to structure XV, wherein R 9 and R 10 are absent, R 11 is propyl, R 7 , R 12 , R 13 , and R 14 are H, Z 1 to Z 4 are oxygen and X is chloride.
  • R 1 , R 2 , R 3 , and R 4 can be the same or different and can be independently H or C 1 to C 32 aliphatic.
  • XVII Shown in structure XVII below is another exemplary compound (a cationic cardiolipin variant ester) according to structure XVI, wherein R 9 and R 10 are absent, R 11 is propyl, R 7 , R 12 , R 13 , and R 14 are H, Z 1 to Z 4 are -O-C(O)-, and X is chloride.
  • Cationic cardiolipin molecules according to the present invention can comprise fatty /alkyl chains (e.g., at Ri, R 2 , R 3 , and R 4 ) of varying length and saturation/unsaturation.
  • the length of the fatty acid hydrocarbon chain ranges from about 2 to 32 carbon atoms, however the carbon chain is more typically between about 10 and about 24 carbon atoms (such as between 14 and 20 carbon atoms).
  • Fatty acids typically are classified by the number of double and/or triple bonds in the hydrocarbon chain (i.e., unsaturation).
  • a saturated fatty acid does not contain any double or triple bonds and each carbon in the chain is bound to the maximum number of hydrogen atoms.
  • the degree of unsaturation of a fatty acid depends on the number of double or triple bonds present in the hydrocarbon chain. In this respect, a monounsaturated fatty acid contains one double bond, whereas a polyunsaturated fatty acid contains two or more double bonds (see e.g., Oxford Dictionary of Biochemistry and Molecular Biology, rev. ed., A.D. Smith (ed.) Oxford University Press (2000), and Molecular Biology of the Cell, 3 rd ed., BA. Alberts (ed.) Garland Publishing, New York (1994)).
  • the fatty acid chains of the cationic cardiolipins can also be saturated or unsaturated.
  • Fatty acids that can be used in the present invention range from carbon chain lengths of about Ci to C 32 , e.g., between about C 4 and C 24 , and include tetranoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic (myristic) acid, pentadecanoic acid, hexadecanoic (palmitic) acid, heptadecanoic acid, octadecanoic (stearic) acid, nonadecanoic acid, eicosanoic (arachidic) acid, heneicosanoic acid, docosanoic (behenic) acid
  • the alkyl chain will also range from C 1 to C 32 , preferably from about C 4 to about C 24 .
  • Other fatty acid chains can also be employed as Ri and/or R 2 , R 3 , and/or R 4 substituents.
  • saturated fatty acids such as ethanoic (or acetic) acid, propanoic (or propionic) acid, butanoic (or butyric) acid, hexacosanoic (or cerotic) acid, octacosanoic (or montanic) acid, triacontanoic (or melissic) acid, dotriacontanoic (or lacceroic) acid, tetratriacontanoic (or gheddic) acid, pentatriacontanoic (or ceroplastic) acid, and the like, monoethenoic unsaturated fatty acids such as tra?w-2-butenoic (or crotonic) acid, cis-2- butenioc (or isocrotonic) acid, 2-hexenoic (or isohydrosorbic) acid, 4-decanoic (or obtusilic) acid, 9-decanoic (or ca) acid,
  • the cationic cardiolipin molecules described above can be made in accordance with any suitable method, such as are known in the art.
  • One synthetic method is detailed in PCT International Publication Number WO04/035523, which is herein incorporated in its entirety by reference. All other publications cited herein are also incorporated in their entirety by reference.
  • the liposomes contain other cationic lipids, either in addition to or instead of a cardiolipin analog.
  • other cationic lipids include, without limitation, DMTAP, DPTAP, DOTAP, DC-Choi, TC-Chol, DPIM, CHIM, DORIE, DDAB, DAC-Chol, DOTMA, DOGS, (C18) 2 Gb/>I,N-dioctadecylamido-glycine, DTAB, CPyC, DODAP, DOEPC and amides from lysine, arginine or ornithine and phosphatidylethanolamine and derivatives and mixtures thereof.
  • the liposomes include zwitterionic lipids. These can be any amphipathic or zwitterionic lipid and can include any of the natural or synthetic phospholipids or mono-, di-or triglycerols.
  • the natural phospholipids are typically those from animal and plant sources, such as phosphatidylcholine, phosphatidylethanolamine, plasmalogens, sphingomyelins, phosphatidylserine, phosphatide acids or phosphatidylinositol.
  • amphipathic or zwitterionic lipids include phosphatidylcholines such as DMPC, DSPC, DOPC, OPPC, DPPC, DAPC, POPC, OPPC, egg PC, soy PC, hydrogenated soy PC, and mixtures thereof.
  • the zwitterionic lipids can also include phosphatidylethanolamines such as DOPE, DMPE and DPPE.
  • the amphipathic or zwitterionic lipid of the liposomal composition is DOPC.
  • Sterols include any steroid-based alcohol (having a hydrocarbon (aliphatic) side-chain of 8-10 carbons at the 17-beta position) and a hydroxyl group at the 3 -beta position.
  • They include without limitation, cholesterol, cholesterol derivatives, coprostanol, cholestanol, cholestane, cholesterolhemisuccinate, cholesterol sulfate, sitosterol, campestenol, desmosterol, coprostenol, fucosterol, 22-ketosterol, 20 hydroxysterol, sigmasterol, 22-hydroxycholesterol, 25-hydroxycholesterol, lanosterol, 7-dehydrocholesterol, dihydrocholesterol, 19- hydroxycholesterol, 5 ⁇ cholest-7-en-3 ⁇ -ol, 7-hydroxycholesterol, dpocholesterol, ergosterol and degydroergosterol and derivatives and mixtures thereof, as well as tocopherols (
  • the liposomal mixture can also include a stabilizer.
  • Suitable stabilizers include compounds such as ascorbic acid, tocopherol and deteroxime mesylate.
  • the stabilizer is tocopherol acid succinate.
  • the mixture can also include an isotonicity adjuster. These include sucrose, trehalose, maltose, lactose and glucose. In one embodiment, the stabilizer is sucrose.
  • an absorption enhancer can be added. These include sodium- salicylate-chenodeoxy cholate, sodium deoxycholate, polyoxyethylene 9-lauryl ether, chenodeoxy cholate-deoxycholate and polyoxethylene-9-lauryl ether, monoolein, sodium tauro-24,25-dihydrofusikate, sodium taurodeoxycholate, sodium glycochenodeoxycholate, oleic acid, linoleic acid, and linolenic acid.
  • Polymeric absorption enhancers can also be included, such as polyoxyethylene ethers, polyoxyethylene sorbitan esters, polyoxyethylene 10-lauryl ether, polyoxyethylene 16-lauryl ether and axone (l-dodecylazacycloheptane-2-one).
  • the bcl-2 gene has two promoters designated Pl and P2.
  • Pl from which most bcl-2 mRNA is transcribed is located approximately 1.4 kb upstream of the translation initiation site and P2 is 1.3 kb downstream of Pl .
  • Pl is GC-rich, lacks a TATA box, has many transcription start sites and includes seven consensus binding sites for the SPl transcription factor.
  • P2 includes a CCAAT box and a TATA box and has two different transcription initiation sites. There are multiple NF -.KB recognition sites and an SV40 enhancer-like octamer motif within P2.
  • TGF- ⁇ , c-ki-ras, c-myc, c-erb-2 (Her-2), and c-Ha-ras can also be investigated to find regions to which oligonucleotides could bind based on preferred design criteria.
  • the oligonucleotides can include any oligomer that hybridizes to the upstream regions of the c-ki-ras, c-Ha-ras, c-myc, her-2, TGF-a, or bcl-2 gene.
  • those upstream regions are defined as SEQ ID NO:1 (for her-2, or c-erb-2), SEQ ID NO:282 (for c-ki-ras), SEQ ID NO:462 (for c-Ha-ras), SEQ ID NO:936 (for c-myc), SEQ ID NO:1081 (for TGF-a) and SEQ ID NOs:1249 and 1254 (for bcl-2).
  • oligonucleotides are designed based on preferred design criteria. Such oligonucleotides can then be tested for efficacy using the methods disclosed herein. For example, in some embodiments, the oligonucleotides are methylated on at least one, two or all of the CpG islands. In other embodiments, the oligonucleotides contain no methylation.
  • the present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that oligonucleotides in some embodiments are those that have at least a 50% GC content and at least two GC dinucleotides.
  • the oligonucleotides do not self hybridize.
  • oligonucleotides are designed with at least 1 A or T to minimize self hybridization.
  • commercially available computer programs are used to survey oligonucleotides for the ability to self hybridize.
  • oligonucleotides are at least 10, or 15 nucleotides and no more than 100 nucleotides in length.
  • oligonucleotides are 18-26 nucleotides in length.
  • oligonucleotides comprise the universal protein binding sequences CGCCC and CGCG or the complements thereof.
  • oligonucleotides hybridize to a promoter region of a gene upstream from the TATA box of the promoter. In further embodiments, oligonucleotides are designed to hybridize to regions of the promoter region of an oncogene known to be bound by proteins ⁇ e.g., transcription factors). In some embodiments, oligonucleotide compounds are not completely homologous to other regions of the human genome. The homology of the oligonucleotide compounds of the present invention to other regions of the genome can be determined using available search tools ⁇ e.g., BLAST, available at the Internet site of NCBI). [0218] The present invention is not limited to the oligonucleotides described herein.
  • oligonucleotides may be identified (e.g., using the criteria described above or other criteria).
  • Candidate oligonucleotides may be tested for efficacy using any suitable method. For example, candidate oligonucleotides can be evaluated for their ability to prevent cell proliferation at a variety of concentrations. In some embodiments, oligonucleotides inhibit gene expression or cell proliferation at a low concentration (e.g., less that 20 ⁇ M, or 10 ⁇ M in in vitro assays.).
  • regions within the promoter region of an oncogene are further defined as regions for hybridization of oligonucleotides, hi some embodiments, these regions are referred to as "hot zones.”
  • hot zones are defined based on oligonucleotide compounds that are demonstrated to be effective (see above section on oligonucleotides) and those that are contemplated to be effective based on the criteria for oligonucleotides described above.
  • hot zones encompass 10 bp upstream and downstream of each compound included in each hot zone and have at least one CG or more within an increment of 40 bp further upstream or downstream of each compound, hi further embodiments, hot zones encompass a maximum of 100 bp upstream and downstream of each oligonucleotide compound included in the hot zone, hi additional embodiments, hot zones are defined at beginning regions of each promoter. These hot zones are defined either based on effective sequence(s) or contemplated sequences and have a preferred maximum length of 200 bp. Based on the above described criteria, exemplary hot zones were designed. These hot zones are shown in Table 1.
  • the oligonucleotides can be any oligomer that hybridizes under physiological conditions to the following sequences: SEQ ID NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ ID NO:936, SEQ ID NO.1081, SEQ ID NOs:1249 and/or 1254.
  • the oligonucleotides can be any oligomer that hybridizes under physiological conditions to exemplary hot zones in SEQ ID NO:1, SEQ ID NO.282, SEQ ID NO:462, SEQ ID NO:936, SEQ ID NO:1081 and SEQ ID NO:1249.
  • oligomers include, without limitation, those oligomers listed in SEQ ID NOs 2-281, 283-461, 463-935, 937-1080, 1082-1248, 1250- 1253 and 1267-1477 and the complements thereof, hi another aspect, the oligonucleotides are SEQ ID NOs 2-22, 283-301, 463-503, 937-958, 1082-1109, 1250-1254 and 1270-1477 and the complements thereof. In an embodiment of these aspects, the oligonucleotides are from 15-35 base pairs in length.
  • the oligomer can be any oligomer that hybridizes to SEQ ID NOs: 1249 or 1254. In another aspect, the oligomer can be any oligomer that hybridizes to nucleotides 500-2026, nucleotides 500-1525, nucleotides 800-1225, nucleotides 900-1125, nucleotides 950-1075 or nucleotides 970-1045 of SEQ ID NO: 1249 or the complement thereof. [0223] In one embodiment, the oligomer can be SEQ ID NO:1250, 1251, 1252, 1253, 1267- 1477 or the complement thereof.
  • the oligomer can be SEQ ID NOs 1250, 1251, 1267, 1268, 1276, 1277, 1285, 1286 or the complement thereof.
  • the oligomer can be SEQ ID NOs 1250, 1251, 1289-1358 or the complements thereof.
  • the oligomer can be SEQ ID NO:1250 or 1251.
  • the oligomer has the sequence of the positive strand of the bcl-2 sequence, and thus, binds to the negative strand of the sequence.
  • the oligomers can include mixtures of bcl-2 oligonucleotides.
  • the oligomer can include multiple oligonucleotides each of which hybridizes to different parts of SEQ ID NOs:1249 and 1254. Oligomers can hybridize to overlapping regions on those sequences or the oligomers may hybridize to non-overlapping regions. In other embodiments, oligomers can be SEQ ID NOs:1250, 1251, 1252, 1253, 1267-1477 or the complement thereof, wherein the mixture of bcl-2 oligomers comprises oligomers of at least 2 different sequences.
  • the oligomer can include a mixture of oligomers, each of which hybridizes to a regulatory region of different genes.
  • the oligomer can include a first oligomer that hybridizes to SEQ ID NO: 1249 or 1254 and second oligomer that hybridizes to a regulatory region of a second gene.
  • the oligomer includes an oligomer of SEQ ID NOs 1250-1254 and 1267-1477 or the complements thereof, and an oligomer that hybridizes to SEQ ID NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ ID NO:936, or SEQ ID NO:1081 or the complement thereof.
  • the oligomer includes SEQ ID NO 1250 or 1251 or the complement thereof and an oligomer that hybridizes to SEQ ID NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ ID NO:936, or SEQ ID NO:1081 or the complement thereof.
  • the oligomer includes SEQ ID NO:1250 or 1251 or the complement thereof and any of SEQ ID NOs 2-281, 283-461, 463-935, 937-1080 and 1082-1248, or the complement thereof.
  • the present invention provides oligonucleotide therapeutics that are methylated at specific sites.
  • the present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that one mechanism for the regulation of gene activity is methylation of cytosine residues in DNA.
  • 5-methylcytosine (5-MeC) is the only naturally occurring modified base detected in DNA (Ehrlick et al, Science 212:1350-1357 (1981)).
  • 5-methylcytosine 5-MeC
  • hypomethylation at specific sites or in specific regions in a number of genes is correlated with active transcription (Doerfler, Annu. Rev. Biochem.
  • DNA methylation in vitro can prevent efficient transcription of genes in a cell-free system or transient expression of transfected genes. Methylation of C residues in some specific cis-regulatory regions can also block or enhance binding of transcriptional factors or repressors (Doerfler, supra; Christman, supra; Cedar, Cell 34:5503-5513 (1988); Tate et al, Curr. Opin. Genet. Dev. 3:225-231 [1993]; Christman et al, Virus Strategies, eds.
  • hypomethylation of DNA is an early event in development of colon cancer (Goetz et al, Science 228:187-290 [1985]). Interference with methylation in vivo can lead to tumor formation. Feeding of methylation inhibitors such as L- methionine or 5-azacytodine or severe deficiency of 5-adenosine methionine through feeding of a diet depleted of lipotropes has been reported to induce formation of liver tumors in rats (Wainfan et al, Cancer Res. 52:207 ls-2077s [1992]).
  • the present invention thus takes advantage of this naturally occurring phenomena, to provide compositions and methods for site specific methylation of specific gene promoters, thereby preventing transcription and hence translation of certain genes.
  • the present invention provides methods and compositions for upregulating the expression of a gene of interest (e.g., a tumor suppressor gene) by altering the gene's methylation patterns.
  • a gene of interest e.g., a tumor suppressor gene
  • the present invention is not limited to the use of methylated oligonucleotides. Indeed, the use of non-methylated oligonucleotides for the inhibition of gene expression is specifically contemplated by the present invention.
  • Example 8 Experiments conducted during the course of development of the present invention (See e.g., Example 8) demonstrated that an unmethylated oligonucleotide targeted toward Bcl-2 inhibited the growth of lymphoma cells to a level that was comparable to that of a methylated oligonucleotide.
  • the oligonucleotides can be in a naturally occurring state, and can also contain modifications or substitutions in the nucleobases, the sugar moiety and/or in the internucleoside linkage.
  • Nucleobases comprise naturally occurring nucleobases as well as non-naturally occurring nucleobases.
  • Illustrative examples of such nucleobases include without limitation adenine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine, guanine, thymine, uracil, 5-bromouracil, 5-propynyluracil, 5-propynylcytosine, 5-propyny-6-fluoroluracil, 5- methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 8-azaguanine, 8-azaadenine, 7-propyne-7- deazaadenine, 7-propyne-7-deazaguanine, 2-chloro-6-aminopurine, 4-acety
  • Oligonucleotides can also have sugars other than ribose and deoxy ribose, including arabinofuranose (described in International Publication number WO 99/67378, which is herein incorporated by reference), xyloarabinofuranose (described in U.S. Patent Nos 6,316,612 and 6,489465, which are herein incorporated by reference), ⁇ -threofuranose (Sch ⁇ ning, et al. (2000) Science, 290, 1347-51, which is herein incorporated by reference) and L-ribofuranose.
  • Sugar mimetics can replace the sugar in the nucleotides. They include cyclohexene (Wang et al.(2000) J. Am. Chem.
  • the oligonucleotides can also include "locked nucleic acids" or LNAs.
  • the LNAs can be bicyclic, tricyclic or polycyclic. LNAs include a number of different monomers, one of which is depicted in Formula XVIII.
  • B constitutes a nucleobase
  • Z* is selected from an internucleoside linkage and a terminal group
  • Z is selected from a bond to the internucleoside linkage of a preceding nucleotide/nucleoside and a terminal group, provided that only one of Z and Z* can be a terminal group;
  • an LNA or LNA* nucleotide can also include "locked nucleic acids" with other furanose or other 5 or 6-membered rings and/or with a different monomer formulation, including 2'-7,3' linked and 3 '-7,4' linked, l'-7,3 linked, 1'- 7,4' linked, 3'-7,5' linked, T-Y, 5'linked, l '-7,2' linked bicyclonucleosides and others.
  • LNA oligonucleotides and LNA nucleotides are generally described in International Publication No. WO 99/14226 and subsequent applications; International Publication Nos. WO 00/56746, WO 00/56748, WO 00/66604, WO 01/25248, WO 02/28875, WO 02/094250, WO 03/006475; U.S. Patent Nos.
  • LNA oligonucleotides and LNA analogue oligonucleotides are commercially available from, for example, Proligo LLC 6200 Lookout Road, Boulder, CO 80301 USA.
  • the nucleotide derivatives can include nucleotides containing one of the following at the 2' sugar position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- orN-alkynyl; or O-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C ⁇ to Cio alk y J or C 2 to Cio alkenyl and alkynyl, O[(CH 2 ) n O] m CH3, O(CH2) n OCH3, O(CH 2 ) n NH2, O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )]2, where n and m are from 1 to about 10, Ci to Cio lower alkyl, substituted lower alkyl, substitute
  • the oligonucleotides have non-natural intemucleoside linkages.
  • oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified oligonucleotides that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides.
  • Some modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • both the sugar and the internucleoside linkage (i.e., the backbone) of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science 254:1497 (1991).
  • oligonucleotides of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH 2 -, -NH-O-CH 2 -, -CH 2 -N(CH 3 )-O-CH 2 - [known as a methylene (methylimino) or MMI backbone], -CH 2 -O-N(CH 3 )-CH 2 -, ⁇ CH 2 -N(CH 3 )-N(CH 3 )-CH 2 -, and -0-N(CH 3 )- CH 2 -CH 2 - [wherein the native phosphodiester backbone is represented as -0-P-O-CH 2 -] of the above referenced U.S.
  • Oligonucleotides can also have a morpholino backbone structure of the above-referenced U.S. Pat. No. 5,034,506.
  • the oligonucleotides have a phosphorothioate backbone having the following general structure.
  • Another modification involves adding additional nucleotides to the 3' and/or 5' ends of the DNAi oligonucleotides.
  • the 3' and 5' tails can comprise any nucleotide and can be as short as one nucleotide and as long as 20 nucleotides.
  • the liposome formulations of the present invention 1) exhibit low toxicity; 2) can sequester high concentrations of oligomers e.g., the efficiency of sequestering the oligonucleotides associated with the amphoteric liposomes is greater than 90%; 3) are stable under various conditions; 4) can be optimized for delivery to animals, such as by adjusting the ratio of lipid to sequestered oligonucleotides to about 100 or greater (e.g., in the range between 100 and 180, such as about 144); and 5) can be produced with an average amphoteric liposome size, e.g., in the range from 50 to 500 ⁇ m, from 80 to 300 ⁇ m , or from 90 to 200 ⁇ m, such as about 144.
  • the cationic liposomes can include one or more bcl-2 oligonucloeitides and/or other oncogene oligonucleotides, cationic lipids, zwitterionic lipids, one or more sterols, and optionally a stabilizer and/or an isotonicity adjuster.
  • any combination of cationic, and zwitterionic lipids and sterols can be used to form liposomes provided that the resulting liposome exhibits stability, has low toxicity, sequesters an ample quantity of oligonucleotides, e.g., with an efficiency of at least about 50%, and provides adjustment of the sequestered oligonucleotide to lipid ratio to about 100 or greater.
  • the molar ratio of the cationic lipid species to the zwitterionic and neutral lipid species is typically between 9/1 and 1/9.
  • the concentration of total lipids, (including cationic and zwitterionic lipids and sterols) in the liposomes is from about 20 to about 40 mg/mL of the lipid mixture (e.g., about 30 mg/mL).
  • the liposomal mixture comprises about 1.4 to about 2.8 mg/mL of a cationic lipid (e.g., 2 mg/mL), about 12.0 to about 22.0 mg/mL of a zwitterionic lipid (e.g., from about 14.9 to about 18.9 mg/mL or about 16 mg/niL), and about 4.0 to about 5.2 mg/mL of a sterol (e.g., about 4.4 to about 4.8, such as 4.6 mg/mL).
  • a cationic lipid e.g., 2 mg/mL
  • a zwitterionic lipid e.g., from about 14.9 to about 18.9 mg/mL or about 16 mg/niL
  • a sterol e.g., about 4.4 to about 4.8, such as 4.6 mg/mL
  • Specific mixtures of cationic and zwitterionic lipids and sterols for sequestering an oligonucleotide, such as SEQ ID NO: 1250 or 1251, include the cationic lipid (R)-PLC-2, the zwitterionic lipid DOPC and the sterol cholesterol. Additionally, specific ratios of the cationic lipids/zwitterionic lipids/sterols lipids can be, e.g., 9-36 mole % of a cationic cardiolipin, 36-72 mole % of a zwitterionic lipid, and 9-36 mole % of a sterol.
  • the liposomal mixture comprises 9-36 mole % (e.g., 20 mole %) of (R)-PLC-2, 36-72 mole % (e.g., 52 mole %) of DOPC, and 9-36 mole % (e.g., 28 mole %) of cholesterol.
  • the liposomal mixture comprises 50-60 mole % (e.g., 54.0 mole %) of DOPC, 10-20 mole% (e.g., 16.0 mole %) of (R)-PLC-2, and 20-40 mole % (e.g., 30 mole %) of cholesterol.
  • the oligonucleotide contributes to the charge ratio of the liposomes, which is important for stability. In one embodiment, the charge ratio is between 1.5 and 2.5 (e.g., at 2.0).
  • the pH of the liposomes is also important for stability. The pH of the liposomal mixture can range from 5.1 to 7.9. In one embodiment, the pH ranges from 6.8 to 7.8 (e.g., 7.2 or 7.6).
  • the diameter of the liposomes is between 50 and 500 ⁇ m (e.g., between 50 and 300 ⁇ m or between 50 and 180 ⁇ m).
  • the liposomal mixture comprises a bcl-2 oligonucleotide, the cationic lipid (R)-PLC-2, the zwitterionic lipid DOPC, the sterol cholesterol, the stabilizer tocopherol acid succinate and the isotonicity adjuster sucrose.
  • the pH is 7.6 and the size is 150 ⁇ m.
  • Cationic liposomes of the invention can be prepared by standard methods for preparing and sizing liposomes known to those skilled in the art. These include hydration of lipid films and powders, solvent injection and reverse-phase evaporation. Often multilamellar vesicles will form spontaneously when amphiphilic lipids are hydrated, whereas the formation of small unilamellar vesicles usually requires a process involving substantial energy input, such as ultrasonication, high pressure homogenization, injection of lipid solutions in ethanol into a water phase containing the cargo to be sequestered and/or extrusion through filters or membranes of defined pore size. Methods for preparing and characterizing liposomes have been described, for example, by S. Vemuri et al. (Preparation and characterization of liposomes as therapeutic delivery systems: a review. Pharm Acta HeIv. 1995, 70(2):95-l 11), which is incorporated herein by reference.
  • the lipids e.g., colipids that include DOPC or (R)-PLC-2) and optionally a stabilizer (e.g., tocopherol acid succinate) can be dissolved or dispersed in a suitable solvent or combination of solvents to give a first mixture.
  • suitable solvents include any non-polar or slightly polar solvent, such as t-butanol, ethanol, methanol, chloroform or acetone.
  • a solution of oligonucleotides optionally containing an isotonicity adjuster e.g., sucrose, trehalose, maltose, lactose, or glucose is next added to the lipid mixture to form a liposomal dispersion.
  • a solution of the oligonucleotide maybe contacted with an excipient at a neutral pH, thereby resulting in a passive loading procedure of a certain percentage of the solution.
  • concentrations of the excipient ranging from about 50 mM to about 150 mM, is one method to achieve substantial encapsulation of the active agent.
  • Excipients include substances that can initiate or facilitate loading of DNAi oligonucleotides.
  • excipients include, without limitation, acid, sodium or ammonium forms of monovalent aniond such as chloride, acetate, lactobionate and formate; divalent anions such as aspartate, succinate and sulfate; and trivalent ions such as citrate and phosphate.
  • a shearing force is applied to the aqueous dispersion of the DNAi-oligonucleotide lipid mixture.
  • the shearing force can be applied by sonication, using a microfluidizing apparatus such as a homogenizer or French press, injection, freezing and thawing, dialyzing away a detergent solution from lipids, extrusion through filters, or other known methods used to prepare liposomes.
  • the size of the liposomes can be controlled using a variety of known techniques, including the duration of shearing force.
  • Unentrapped oligomers are removed from the liposome dispersion by buffer exchange using dialysis, size exclusion chromatography (e.g., Sephadex G-50 resin), ultrafiltration (100,000-300,000 molecular weight cutoff), or centrifugation.
  • size exclusion chromatography e.g., Sephadex G-50 resin
  • ultrafiltration 100,000-300,000 molecular weight cutoff
  • the method includes adding an oligomer that comprises the sequence of PNT-100, e.g., to give the first mixture containing about 1-4 mg/mL (e.g., 2 mg/mL) of PNT-100 oligomer.
  • the cationic liposomes system thus prepared has a concentration of total lipids of about 10 to 40 mg/mL (e.g., 30.0 mg/ml).
  • the first mixture thus prepared includes from about 4.40 mg/mL to about 4.8 mg/mL of cholesterol.
  • the first dispersion or mixture includes from about 12 to 24 mg/niL (e.g., from about 14.9 mg/mL to about 18.9 mg/niL) of DOPC; or from about 5.4 to about 12 (e.g., from about 6.3 mg/mL to about 10.6 mg/mL) of (R)-PLC-2.
  • the second mixture thus prepared has a pH of between about 4.4 and about 8.6 (e.g., between 5.1 and about 7.9).
  • a liposomal delivery system thus prepared contains from about 12.0 mg/mL to about 24 mg/mL (e.g., from about 14.9 mg/mL to about 18.9 mg/mL) of DOPC, from about 3.8 mg/mL to about 5.2 mg/mL (e.g., from about 4.4 mg/mL to about 4.8 mg/mL) of cholesterol, from about 5.4 mg/mL to about 12.0 mg/mL (e.g., from about 6.3 mg/mL to about 10.6 mg/mL) of (R)-P LC-2, tocopherol acid succinate, and sucrose.
  • the liposomal delivery system has a pH of between about 5.1 and about 7.9.
  • oligonucleotide compounds of the present invention maybe delivered using any suitable method.
  • naked DNA is administered.
  • lipofection is utilized for the delivery of nucleic acids to a subject.
  • oligonucleotides are modified with phosphothioates for delivery (See e.g., U.S. Patent 6,169,177, herein incorporated by reference).
  • nucleic acids for delivery are compacted to aid in their uptake (See e.g., U.S. Patents 6,008,366, 6,383,811 herein incorporated by reference).
  • compacted nucleic acids are targeted to a particular cell type (e.g., cancer cell) via a target cell binding moiety (See e.g., U.S. Patents 5,844,107, 6,077,835, each of which is herein incorporated by reference).
  • oligonucleotides are conjugated to other compounds to aid in their delivery.
  • nucleic acids are conjugated to polyethylene glycol to aid in delivery (See e.g., U.S. Patents 6,177,274, 6,287,591, 6,447,752, 6,447,753, and 6,440,743, each of which is herein incorporated by reference).
  • oligonucleotides are conjugated to protected graft copolymers, which are chargeable" drug nano-carriers (Pharmaln), described in U.S. Patent Number 7.138,105, and U.S. publication numbers 2006/093660 and 2006/0239924, which are incorporated herein by reference.
  • oligonucleotides are conjugated to nanoparticles (e.g., NanoMed Pharmaceuticals; Kalamazoo, MI).
  • oligonucleotides are associated with dendrimers. Dendrimers are synthetic macromolecules with highly branched molecular structures.
  • dendrimeric structures are cationic polymers such as starburst polyamidoamine (PAMAM), one of which, SuperFect ® , is available from Qiagen (Valencia, CA).
  • PAMAM starburst polyamidoamine
  • SuperFect ® is available from Qiagen (Valencia, CA).
  • Other dendrimers include polyester dendrimers described by Gillies, et al., MoI.
  • amphiphilic dendrimers described by Joester, et al., Angew Chem Int. Ed. Engl, 42: 1486-90, 2003, which is incorporated herein by reference; polyethylene glycol star like conjugates, described by Liu et al., Polym Chem, 37:3492-3503, 1999, which is incorporated herein by reference; cationic phosphorus-containing dendrimers described by Loup, et al., Chem Eur J, 5:3644-50, 1999, which is incorporated herein by reference; poly(L-lysine) dendrimers, described by Ohasaki, et al., Bioconjug Chem, 13:510- 17, 2002, which is incorporated herein by reference and amphipathic asymmetric dendrimers, described by Shah, et al., Int.
  • Dendrimers complex with nucleic acids as do other cationic polymers with high charge density. In general, the dendrimer-nucleic acid interaction is based on electrostatic interactions.
  • Dendrimers can be conjugated with other molecules, such as cyclodextrins to increase efficiency of systemic delivery of dendrimer-nucleic acid complexes.
  • cyclodextrins to increase efficiency of systemic delivery of dendrimer-nucleic acid complexes.
  • oligonucleotides are sequestered in polymer vesicles.
  • Polymer vesicles can be made from a number of different materials, but in general are formed from block copolymers, for example, polystyrene 4 o- ⁇ oly(isocyano-L-alanine-L-alanine) m .
  • block copolymers for example, polystyrene 4 o- ⁇ oly(isocyano-L-alanine-L-alanine) m .
  • Copolymer vesicles are formed from a number of molecules, including, without limitation, polyacrylic acid-polystyrene, nonionic polyethyleneoxide-polybutadiene, the triblock (polyethyleneoxide)5-(poly[propyleneoxide]) 6 8- (polyethyleneoxide) 5 , polyethyleneoxide-poly(propylenesulfide), polyethyleneoxide- polylactide, and polyethylene glycol-polylysine.
  • copolymers particularly those of either amphiphilic or oppositely charged copolymers, including polystyrene 4 o-poly(isocyano-L- alanine-L-alanine) m , self assemble into vesicles in aqueous conditions.
  • Oligonucleotides can be loaded into the polymer vesicles using several methods.
  • a third method involves dissolving both the oligonucleotides and copolymer in a water/tert-butanol mixture and subsequent lyophilization of the solvents.
  • the oligonucleotide-loaded vesicles are formed spontaneously when the lyophilized oligonucleotide-copolymer is reconstituted in an injectable vehicle.
  • Polymer vesicles can be targeted to specific cells by tethering a ligand to the outer shell of vesicles by post modification of a copolymer with a bifunctional spacer molecule or by the direct synthesis of heterobifunctional block copolymers.
  • oligonucleotides are enclosed in liposomes or micelles to aid in delivery.
  • liposomes or micelles In addition to the cationic liposome formulations described herein, other liposomal formulations can be used. (See e.g., U.S. Patents 6,458,382, 6,429,200; U.S Patent Publications 2003/0099697, 2004/0120997, 2004/0131666, 2005/0164963, and PCT International Publication WO 06/048329, each of which is herein incorporated by reference).
  • Liposomes include, without limitation, cardiolipin based cationic liposomes described herein as well as other cardiolipin based liposomes (e.g., NeoPhectin, available from NeoPharm, Forest Lake, IL) and pH sensitive liposomes.
  • cardiolipin based cationic liposomes described herein as well as other cardiolipin based liposomes (e.g., NeoPhectin, available from NeoPharm, Forest Lake, IL) and pH sensitive liposomes.
  • NeoPhectin is utilized as the liposomal delivery vehicle.
  • the NeoPhectin is formulated with the oligonucleotide so as to reduce free NeoPhectin.
  • NeoPhectin is present at a charge ratio 6:1 or less (e.g., 5:1, and 4:1) of NeoPhectin to oligonucleotide.
  • lipids particularly phospholipids that comprise some liposomes, are conjugated to polyethylene glycol or a derivative thereof, to increase the time that the liposomes circulate in the blood after intravenous injection.
  • Such liposomes termed "stealth liposomes" are able to avoid the reticuloentothelial system (RES), resulting in half lives of more than 24 hours in some cases.
  • the phospholipids in liposomes are conjugated to polyethylene glycol-diorthoester molecules, as described in Li, W., et al., J. Gene Med., 7:67-79, 2005.
  • the PEG-liposomes are targeted to specific cell receptors.
  • haloperidol conjugated at the distal end of a PEG-linked phospholipids in a cationic liposome targeted sigma receptors that are overexpressed on some cancer cells as described in Mukherjee, et al., J. Biol. Chem., 280, 15619-27, 2005, which is incorporated herein by reference.
  • Anisamide conjugated to PEG-linked phospholipids in liposomes also targets the sigma receptor. (Banerjee, et al., Int. J. Cancer, 112, 693-700, 2004, which is incorporated herein by reference.)
  • oligonucleotides can be sequestered in hybrid liposome- copolymer vesicles, as described in Ruysschaert, et.al., J. Am. Chem. Soc, 127, 6242-47, 2005, which is incorporated herein by reference.
  • amphiphilic triblock copolymers including poly(2-methyloxazoline)-block-poly(dimethylsiloxan)-block-poly(2- methyloxazoline) can interact with lipids, including phospholipids to form hybrid liposome- copolymer vesicles.
  • oligonucleotides are complexed with additional polymers to aid in delivery (See e.g., U.S. Patents 6,379,966, 6,339,067, 5,744,335; each of which is herein incorporated by reference.
  • additional polymers See e.g., U.S. Patents 6,379,966, 6,339,067, 5,744,335; each of which is herein incorporated by reference.
  • polymers of N-2-hydroxypropyl methylacrylamide are described in U.S. patent publication number 2006/0014695, which is incorporated herein by reference.
  • Similar cationic polymers are described in PCT International Patent Publication number WO 03/066054 and U.S. patent publication number 2006/0051315, both of which are incorporated herein by reference.
  • Other polymers are described by Intradigm Corp., Rockville, MD).
  • the controlled high pressure delivery system developed by Mirus is utilized for delivery of oligonucleotides.
  • the delivery system is described in U.S. patent number 6,379,966, which is incorporated herein by reference.
  • compositions of the present invention are provided in combination with existing therapies. In other embodiments, two or more compounds of the present invention are provided in combination. In some embodiments, the compounds of the present invention are provided in combination with known cancer chemotherapy agents. The present invention is not limited to a particular chemotherapy agent.
  • antineoplastic (e.g., anticancer) agents are contemplated for use in certain embodiments of the present invention.
  • Anticancer agents suitable for use with the present invention include, but are not limited to, agents that induce apoptosis, agents that inhibit adenosine deaminase function, inhibit pyrimidine biosynthesis, inhibit purine ring biosynthesis, inhibit nucleotide interconversions, inhibit ribonucleotide reductase, inhibit thymidine monophosphate (TMP) synthesis, inhibit dihydrofolate reduction, inhibit DNA synthesis, form adducts with DNA, damage DNA, inhibit DNA repair, intercalate with DNA, deaminate asparagines, inhibit RNA synthesis, inhibit protein synthesis or stability, inhibit microtubule synthesis or function, and the like.
  • any oncolytic agent that is routinely used in a cancer therapy context finds use in the compositions and methods of the present invention.
  • the U.S. Food and Drug Administration maintains a formulary of oncolytic agents approved for use in the United States. International counterpart agencies to the U.S.F.D.A. maintain similar formularies.
  • Chemotherapy agents of the present invention can include any suitable chemotherapy drug or combinations of chemotherapy drugs (e.g., a cocktail).
  • exemplary chemotherapy agents include, without limitation, alkylating agents, platinums, anti-metabolites, anthracyclines, taxanes, camptothecins, nitrosoureas, EGFR inhibitors, antibiotics, HER2/neu inhibitors, angiogenesis inhibitors, kinase inhibitors, proteaosome inhibitors, immunotherapies, hormone therapies, photodynamic therapies, cancer vaccines, histone deacetylase inhibitors, sphingolipid modulators, oligomers, other unclassified chemotherapy drugs and combinations thereof.
  • Alkylating agents are chemotherapy agents that are thought to attack the negatively charged sites on the DNA (e.g., the oxygen, nitrogen, phosphorous and sulfur atoms) and bind to the DNA thus altering replication, transcription and even base pairing. It is also believed that alkylation of the DNA also leads to DNA strand breaks and DNA strand cross-linking. By altering DNA in this manner, cellular activity is effectively stopped and the cancer cell will die.
  • Common alkylating agents include, without limitation, Procarbazine, Ifosphamide, Cyclophosphamide, Melphalan, Chlorambucil, Decarbazine, Busulfan, Thiotepa, and the like. Alkylating agents such as those mentioned above can be used in combination with one or more other alkylating agents and/or with one or more chemotherapy agents of a different class(es).
  • Platinum chemotherapy agents are believed to inhibit DNA synthesis, transcription and function by cross-linking DNA subunits. (The cross-linking can happen either between two strands or within one strand of DNA.) Common platinum chemotherapy agents include, without limitation, Cisplatin, Carboplatin, Oxaliplatin, Eloxatin, and the like. Platinum chemotherapy agents such as those mentioned above can be used in combination with one or more other platinums and/or with one or more chemotherapy agents of a different class(es).
  • Anti-metabolite chemotherapy agents are believed to interfere with normal metabolic pathways, including those necessary for making new DNA.
  • Common anti-metabolites include, without limitation, Methotraxate, 5-Fluorouracil (e.g., Capecitabine), Gemcitabine (2'-deoxy- 2',2'-difluorocytidine monohydrochloride ( ⁇ -isomer), Eli Lilly), 6-mercaptopurine, 6- thioguanine, fludarabine, Cladribine, Cytarabine, tegafur, raltitrexed, cytosine arabinoside, and the like.
  • Gallium nitrate is another anti-metabolite that inhibits ribonucleotides reductase.
  • Anti-metabolites such as those mentioned above can be used in combination with one or more other anti-metabolites and/or with one or more chemotherapy agents of a different class(es).
  • Anthracyclines are believed to promote the formation of free oxygen radicals. These radicals result in DNA strand breaks and subsequent inhibition of DNA synthesis and function. Anthracyclines are also thought to inhibit the enzyme topoisomerase by forming a complex with the enzyme and DNA. Common anthracyclines include, without limitation, Daunorubicin, Doxorubicin, Idarubicin, Epirubicin, Mitoxantrone, adriamycin, bleomycin, mitomycin-C, dactinomycin, mithramycin and the like. Anthracyclines such as those mentioned above can be used in combination with one or more other anthracyclines and/or with one or more chemotherapy agents of a different class(es).
  • Taxanes are believed to bind with high affinity to the microtubules during the M phase of the cell cycle and inhibit their normal function.
  • Common taxanes include, without limitation, Paclitaxel, Docetaxel, Taxotere, Taxol, taxasm, 7-epipaclitaxel, t-acetyl paclitaxel, 10-desacetyl-paclitaxel, lO-desacetyl-7-epipaclitaxel, 7-xylosylpaclitaxel, lO-desacetyl-7- epipaclitaxel, 7-N-N-dimethylglycylpaclitaxel, 7-L-alanylpaclitaxel and the like. Taxanes such as those mentioned above can be used in combination with one or more other taxanes and/or with one or more chemotherapy agents of a different class(es).
  • Camptothecins are thought to complex with topoisomerase and DNA resulting in the inhibition and function of this enzyme. It is further believed that the presence of topoisomerase is required for on-going DNA synthesis.
  • Common camptothecins include, without limitation, Irinotecan, Topotecan, Etoposide, vinca alkaloids (e.g., Vincristine, Vinblastine or Vinorelbine), amsacrine, teniposide and the like. Camptothecins such as those mentioned above can be used in combination with one or more other camptothecins and/or with one or more chemotherapy agents of a different class(es).
  • Nitrosoureas are believed to inhibit changes necessary for DNA repair. Common nitrosoureas include, without limitation, Carmustine (BCNU), Lomustine (CCNU), semustine and the like. Nitrosoureas such as those mentioned above can be used in combination with one or more other nitrosoureas and/or with one or more chemotherapy agents of a different class(es).
  • EGFR i.e., epidermal growth factor receptor
  • EGFR inhibitors include molecules that inhibit the function or production of one or more EGFRs. They include small molecule inhibitors of EGFRs, antibodies to EGFRs, antisense oligomers, RNAi inhibitors and other oligomers that reduce the expression of EGFRs.
  • Common EGFR inhibitors include, without limitation, Gefitinib, Erlotinib (Tarceva ® ), Cetuximab (Erbitux ® ), panitumumab (VectibixTM' Amgen) lapatinib (GlaxoSmithKline), CI1O33 or PDl 83805 or Canternib (6-acrylamide-N-(3 -chloro-4-flurorphenyl)-7-(3 -morpholinopropoxy)quinazolin-4- amine, Pfizer), and the like.
  • inhibitors include PKI-166 (4-[(lR)-l-phenylethylamino]- 6-(4-hydroxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine, Novartis), CL-387785 (N-[4-(3- bromoanilino)quinazolin-6-yl]but-2-ynamide), EKB-569 (4-(3-chloro-4-fluroranilino)-3- cyano-6-(4-dimethylaminobut2(E)-enamido)-7-ethozyquinoline, Wyeth), lapatinib (GW2016, GlaxoSmithKline), EKB509 (Wyeth), Panitumumab (ABX-EGF, Abgenix), matuzumab (EMD 72000, Merck) and the monoclonal antibody RH3 (New York Medical).
  • EGFR inhibitors such as those mentioned above can be used in combination with one or more other EGFR inhibitors
  • Antibiotics are thought to promote the formation of free oxygen radicals that result in DNA breaks leading to cancer cell death.
  • Common antibiotics include, without limitation, Bleomycin and rapamycin and the like.
  • the macrolide fungicide rapamycin also called RAP, Rapamune and Sirolimus
  • FKLBP 12 immunophilin FK506 binding protein 12
  • mTOR mammalian target of rapamycin
  • Rapamycin macrolides include naturally occurring forms of rapamycin as well as rapamycin analogs and derivatives that target and inhibit mTOR.
  • rapamycin macrolides include, without limitation, temsirolimus (CCI-779, Wyeth)), Everolimus and ABT-578.
  • Antibiotics such as those mentioned above can be used in combination with one or more other antibiotics and/or with one or more chemotherapy agents of a different class(es).
  • HER2/neu Inhibitors are believed to block the HER2 receptor and prevent the cascade of reactions necessary for tumor survival.
  • Her2 inhibitors include molecules that inhibit the function or production of Her2. They include small molecule inhibitors of Her2, antibodies to Her2, antisense oligomers, RNAi inhibitors and other oligomers that reduce the expression of tyrosine kinases. Common HER2/neu inhibitors include, without limitation, Trastuzumab (Herceptin®, Genentech) and the like.
  • Her2/neu inhibitors include bispecific antibodies MDX-210(FC ⁇ Rl-Her2/neu) and MDX-447 (Medarex), pertuzumab (rhuMAb 2C4, Genentech), HER2/neu inhibitors such as those mentioned above can be used in combination with one or more other HER2/neu inhibitors and/or with one or more chemotherapy agents of a different class(es).
  • Angiogenesis inhibitors are believed to inhibit vascular endothelial growth factor , i.e. VEGF, thereby inhibiting the formation of new blood vessels necessary for tumor life.
  • VEGF inhibitors include molecules that inhibit the function or production of one or more VEGFs. They include small molecule inhibitors of VEGF, antibodies to VEGF, antisense oligomers, RNAi inhibitors and other oligomers that reduce the expression of tyrosine kinases. Common angiogenesis inhibitors include, without limitation, Bevacizumab (Avastin ® , Genentech).
  • angiogenesis inhibitors include, without limitation, ZD6474 (AstraZeneca), Bay-43-9006, sorafenib (Nexavar, Bayer), semaxamib (SU5416, Pharmacia), SU6668 (Pharmacia), ZD4190 (N-(4-bromo-2-fluoro ⁇ henyl)-6-methoxy-7-[2-(lF-l,2,3-triazol-l- yl)ethoxy]quinazolin-4-amine, Astra Zeneca), ZactimaTM (ZD6474, N-(4-b ⁇ omo-2- fluorophenyl)-6-methoxy-7-[2-(lH- 1 ,2,3-triazol- 1 -yl)ethoxy] quinazolin-4-arnine, Astra Zeneca), Vatalanib, (PTK787, Novartis), the monoclonal antibody IMC-ICl 1 (Imclone) and the like.
  • Aurora kinase inhibitors include, without limitation, compounds such as 4-(4-N benzoylamino)aniline)-6-methyxy-7-(3-(l-morpholino)propoxy)quinazoline (ZM447439, Ditchf ⁇ eld et al., J. Cell. Biol, 161:267-80 (2003)) and Hesperadin (Haaf et al, J. Cell Biol., 161: 281-94 (2003)).
  • SRC/Abl kinase inhibitors include, without limitation, AZD0530 (4-(6-chloro-2,3-methylenedioxyanilino)-7-[2- (4-methylpiperazin-l-yl)ethoxy]-5-tetrahycropyran-4-yloxyquinazoline).
  • Tyrosine kinase inhibitors include molecules that inhibit the function or production of one or more tyrosine kinases.
  • CEP-701 and CEP-751 act as tyrosine kinase inhibitors.
  • Imatinib mesylate is a tyrosine kinase inhibitor that inhibits bcr-abl by binding to the ATP binding site of bcr-abl and competitively inhibiting the enzyme activity of the protein.
  • imatinib is quite selective for bcr-abl, it does also inhibit other targets such as c-kit and PDGF-R.
  • FLT-3 inhibitors include, without limitation, tandutinib (MLN518, Millenium), Sutent (SU11248, 5- [5-fluoro-2-oxo-l,2- dihydroindol-(3Z)-ylidenemethyl]-2, 4-dimethyl-lH- pyrrole-3-carboxylic acid [2-diethylaminoethyl] amide, Pfizer), midostaurin (4'-N-Benzoyl Staurosporine,_Novartis), lefunomide (SUlOl) and the like.
  • MEK inhibitors include, without limitation, 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide) (PDl 84352/CI-1044, Pfizer), PD198306 (Pfizer), PD98059 (2'-amino-3'-methoxyflavone), UO126 (Promega), Ro092210 from fermented microbial extracts (Roche), the resorcyclic acid lactone, L783277, also isolated from microbial extracts (Merck) and the like. Tyrosine kinase inhibitors such as those mentioned above can be used in combination with one or more other tyrosine kinase inhibitors and/or with one or more chemotherapy agents of a different class(es).
  • Proteaosome inhibitors are believed to inhibit the breakdown of some of these proteins that have been marked for destruction. This results in growth arrest or death of the cell.
  • Common proteaosome inhibitors include, without limitation, Bortezomib, ortezomib and the like. Proteaosome inhibitors such as those mentioned above can be used in combination with one or more other proteaosome inhibitors and/or with one or more chemotherapy agents of a different class(es).
  • Immunotherapies are thought to bind to and block specific targets, thereby disrupting the chain of events needed for tumor cell proliferation.
  • Common immunotherapies include, without limitation, Rituximab and other antibodies directed against CD20, Campath-IH and other antibodies directed against CD-50, epratuzmab and other antibodies directed against CD- 22, galiximab and other antibodies directed atainst CD-80, apolizumab HUlDlO and other antibodies directed against HLA-DR, and the like.
  • Radioisotopes can be conjugated to the antibody, resulting in radioimmunotherapy.
  • Two such anti-CD20 products are tositumomab (Bexxar) and ibritumomab (Zevalin).
  • Immunotherapies such as those mentioned above can be used in combination with one or more other immunotherapies and/or with one or more chemotherapy agents of a different class(es).
  • Hormone therapies are thought to block cellular receptors, inhibit the in vivo production of hormones, and/or eliminate or modify hormone receptors on cells, all with the end result of slowing or stopping tumor proliferation.
  • Common hormone therapies include, without limitation, antiestrogens (e.g., tamoxifen, toremifene, fulvestrant, raloxifene, droloxifene, idoxyfene and the like), progestogens) e.g., megestrol acetate and the like) aromatase inhibitors (e.g., Anastrozole, Letrozole, Exemestane, vorazole, exemestane, fadrozole, aminoglutethimide, exemestane, l-methyl-l,4-androstadiene-3,17-dione and the like), anti- androgens (e.g., Bicalutimide, Nilutamide, Flutamide, cy
  • Photodynamic therapies expose a photosensitizing drug to specific wavelengths of light to kill cancer cells.
  • Common photodynamic therapies include, for example, porfimer sodium (e.g., Photofrin ® ) and the like.
  • Photodynamic therapies such as those mentioned above can be used in combination with one or more other photodynamic therapies and/or with one or more chemotherapy agents of a different class(es). 17. Cancer Vaccines
  • Cancer vaccines are thought to utilize whole, inactivated tumor cells, whole proteins, peptide fragments, viral vectors and the like to generate an immune response that targets cancer cells.
  • Common cancer vaccines include, without limitation, modified tumor cells, peptide vaccine, dendritic vaccines, viral vector vaccines, heat shock protein vaccines and the like.
  • Histone deacetylase inhibitors are able to modulate transcriptional activity and consequently, can block angiogenesis and cell cycling, and promote apoptosis and differentiation.
  • Histone deacetylase inhibitors include, without limitation, SAHA (Suberoylanilide hydroxamic acid), depsipeptide (FK288) and analogs, Pivanex (Titan), CI994 (Pfizer), MS275 PXDlOl (CuraGen, TopoTarget) MGCD0103 (MethylGene), LBH589, NVP- LAQ824 (Novartis) and the like and have been used as chemotherapy agents.
  • Histone deacetylase inhibitors such as those mentioned above can be used in combination with one or more other histone deacetylase inhibitors and/or with one or more chemotherapy agents of a different class(es).
  • Ceramide has been shown to induce apoptosis, consequently, exogenous ceramide or a short chain ceramide analog such as N-acetylsphingosine (C 2 -Cer), C 6 -Cer or C 8 -Cer has been used.
  • Other analogs include, without limitation, Cer 1-glucuronide, poly(ethylene glycol)-derivatized ceramides and pegylated ceramides.
  • Modulators that stimulate ceramide synthesis have been used to increase ceramide levels.
  • Compounds that stimulate serine palmitoyltransferase, an enzyme involved in ceramide synthesis include, without limitation, tetrahydrocannabinol (THC) and synthetic analogs and anandamide, a naturally occurring mammalian cannabinoid.
  • THC tetrahydrocannabinol
  • Gemcitabine, retinoic acid and a derivative, fenretinide [N-(4-hycroxyphenyl)retinarnide, (4-HPR)], camptothecin, homocamptothecin, etoposide, paclitaxel, daunorabicin and fludarabine have also been shown to increase ceramide levels.
  • valspodar PSC833, Novartis
  • a non- immunosuppressive non-ephrotoxic analog of cyclosporin and an inhibitor of p-glycoprotein increases ceramide levels.
  • Modulators of sphingomyelinases can increase ceramide levels. They include compounds that lower GSH levels, as GSH inhibits sphingomyelinases. For example, betathine ( ⁇ -alanyl cysteamine disulphide), oxidizes GSH, and has produced good effects in patients with myeloma, melanoma and breast cancer.
  • betathine ⁇ -alanyl cysteamine disulphide
  • COX-2 inhibitors such as celecoxib, ketoconazole, an antifungal agent, doxorubicin, mitoxantrone, D609 (tricyclodecan-9-yl- xanthogenate), dexamethasone, and Ara-C (l-/?-D-arabinofuranosylcytosine) also stimulate sphingomyelinases.
  • the enzyme, GlcCer glucosidase which is available for use in Gaucher' s disease, particularly with retinol or pentanol as glucose acceptors and/or an activator of the enzyme can be used as therapeutic agents.
  • Saposin C and analogs thereof, as well as analogs of the antipsychotic drug, chloropromazine may also be useful.
  • Inhibitors of glucosylceramide synthesis include, without limitation, PDMP (N-[2- hydroxy- 1 -(4-morpholinylmethyl)-2-phenylethyldecanamide]), PMPP (O,L-threo-( 1 -phenyl-2- hexadecanoylamino-3-morpholino-l-propanol), P4 or PPPP (D-threo-l -ph.eny ⁇ -2- palmitoylamino-3 -pyrrolidino- 1 -propanol), ethylenedioxy-P4, 2-decanoylamine-3 - morpholinoprophenone, tamixofen, raloxifene, mifepristone (RU486), N-butyl deoxynojirimycin and anti androgen chemotherapy (bicalutamide + leuprolide acetate).
  • Inhibitors of ceramidase include, without limitation, N-oleoylethanolamine, a truncated form of ceramide, D-MAPP (D-ery ⁇ ro-2-tetradecanoylamino-l -phenyl- 1 -propanol) and the related inhibitor B13 (p-nitro-D-MAPP).
  • Inhibitors of sphingosine kinase also result in increased levels of ceramide.
  • Inhibitors include, without limitation, safingol (L-t/ ⁇ reo-dihydrosphingosine), N,N-dimethyl sphingosine, trimethylsphingosine and analogs and derivatives of sphingosine such as dihydrosphingosine, and myriocin.
  • ceramide levels include, without limitation, miltefosine (hexadecylphosphocholine).
  • Sphingolipid modulators such as those mentioned above, can be used in combination with one or more other sphingolipid modulators and/or with one or more chemotherapy agents of a different class(es).
  • oligonucleotides of the present invention include, without limitation, Genasense (oblimersen, G3139, from Genta), an antisense oligonucleotide that targets bcl-2 and G4460 (LR3001, from Genta) another antisense oligonucleotide that targets c-myb.
  • Genasense oblimersen, G3139, from Genta
  • an antisense oligonucleotide that targets bcl-2 and G4460 LR3001, from Genta
  • Other oligomers include, without limitation, siRNAs, decoys, RNAi oligonucleotides and the like. Oligomers, such as those mentioned above, can be used in combination with one or more other oligomers and/or with one or more chemotherapy agents of a different class(es).
  • Chemotherapy agents can include cocktails of two or more chemotherapy drugs mentioned above.
  • a chemotherapy agent is a cocktail that includes two or more alkylating agents, platinums, anti-metabolites, anthracyclines, taxanes, camptothecins, nitrosoureas, EGFR inhibitors, antibiotics, HER2/neu inhibitors, angiogenesis inhibitors, kinase inhibitors, proteaosome inhibitors, immunotherapies, hormone therapies, photodynamic therapies, cancer vaccines, sphingolipid modulators, oligomers or combinations thereof.
  • radiation therapy is administered in addition to the administration of an oligonucleotide compound.
  • Radiation therapy includes both external and internal radiation therapies.
  • External radiation therapies include directing high-energy rays (e.g., x-rays, gamma rays, and the like) or particles (alpha particles, beta particles, protons, neutrons and the like) at the cancer and the normal tissue surrounding it.
  • the radiation is produced outside the patient's body in a machine called a linear accelerator.
  • External radiation therapies can be combined with chemotherapies, surgery or oligonucleotide compounds.
  • Internal radiation therapies include placing the source of the high-energy rays inside the body, as close as possible to the cancer cells. Internal radiation therapies can be combined with external radiation therapies, chemotherapies or surgery. [0314] Radiation therapy can be administered with chemotherapy simultaneously, concurrently, or separately. Moreover radiation therapy can be administered with surgery simultaneously, concurrently, or separately.
  • cancerous tissue can be excised from a patient using any suitable surgical procedure including, for example, laparoscopy, scalpel, laser, scissors and the like, hi several embodiments, surgery is combined with chemotherapy. In other embodiments, surgery is combined with radiation therapy. In still other embodiments, surgery is combined with both chemotherapy and radiation therapy.
  • the oligonucleotide compounds of the present invention are formulated as pharmaceutical compositions for delivery to a subject as a pharmaceutical.
  • the novel antigen compounds of the present invention find use in the treatment of a variety of disease states and conditions in which it is desirable to inhibit the expression of a gene or the growth of a cell.
  • the compounds are used to treat disease states resulting from uncontrolled cell growth, for example including, but not limited to, cancer.
  • the present invention is not limited to the treatment of a particular cancer.
  • the oligonucleotide compounds of the present invention are suitable for the treatment of a variety of cancers including, but not limited to, breast, colon, lung, stomach, pancreatic, bladder, leukemia and lymphoma.
  • the below discussion provides exemplary, non-limiting examples of formulations and dosages.
  • the present invention further provides pharmaceutical compositions (e.g., comprising the oligonucleotide compounds described above).
  • the pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, intraocular, epidermal and transdermal), oral, via a medical device or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • compositions of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • compositions include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
  • Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention.
  • cationic lipids such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97/30731), also enhance the cellular uptake of oligonucleotides.
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets.
  • oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators.
  • surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Prefered bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro- fusidate, sodium glycodihydrofusidate.
  • DCA chenodeoxycholic acid
  • UDCA ursodeoxychenodeoxycholic acid
  • cholic acid dehydrocholic acid
  • deoxycholic acid deoxycholic acid
  • glucholic acid glycholic acid
  • glycodeoxycholic acid taurocholic acid
  • taurodeoxycholic acid sodium tauro-24,25-dihydro- fusidate, sodium glycodihydrofusidate.
  • Prefered fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1- monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acyl choline, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium).
  • arachidonic acid arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,
  • penetration enhancers for example, fatty acids/salts in combination with bile acids/salts.
  • a particularly prefered combination is the sodium salt of lauric acid, capric acid and UDCA.
  • Further penetration enhancers include polyoxyethylene-9- lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles.
  • Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches.
  • complexing agents include chitosan, N-tiimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.
  • PEG polyethyleneglycol
  • Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and the delivery means, and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models or based on the examples described herein.
  • dosage is from 0.01 ⁇ g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly.
  • dosage is continuous (e.g., intravenously) for a period of from several hours to several days or weeks.
  • treatment is given continuously for a defined period followed by a treatment free period.
  • the pattern of continuous dosing followed by a treatment free period is repeated several times (e.g., until the disease state is diminished).
  • the treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
  • oligonucleotide is administered in maintenance doses, ranging from 0.01 ⁇ g to 100 g, from 1 mg to 50 mg, and from 6 mg to 30 mg per kg of body weight, once or more daily, to once every 20 years.
  • the amphoteric liposomes are useful for treating cells, tissue, or a patient, including an animal (e.g., mice, horses, cats, dogs) and a human, to treat cancer, such as by inhibiting or reducing tumor growth.
  • the mixture is introduced to the animal at a dosage of between 0.01 mg to 100 mg per kg of body weight.
  • the amphoteric liposomes can be introduced to the animal one or more times per day or continuously.
  • the mixture can be administered to the animal via different routes. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, intraocular, epidermal and transdermal), oral or parenteral.
  • Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, intraocular, epidermal and transdermal), oral or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Administration may also be via a medical device.
  • the liposomes may be administered to cultured cells derived from various cancers, including pancreatic cancer, colon cancer, breast cancer, bladder cancer, lung cancer, leukemia, prostate cancer, lymphoma, ovarian cancer or melanoma.
  • the liposomes may also contain a cell targeting component, which may include a ligand for a cell surface or nuclear receptor.
  • lipids are available from commercial sources.
  • Cholesterol is available from Avanti Polar Lipids, Inc, Alabaster, AL; and DOPC can be obtained from Sigma Chemical Company.
  • EXAMPLE 2 Production of Cationic Liposomes Charged with Oligonucleotides
  • the liposomes were produced using an ethanol injection method. Lipids were dissolved in ethanol and a stabilizer was optionally dissolved with the lipids. The lipid mixture was added to a solution of oligonucleotides to form a homogenous liposome dispersion. An isotonicity adjuster was optionally added to the oligonucleotide solution. The dispersion was extruded through stacked polycarbonate membrane filters using a Lipex extruder (Northern Lipids Inc., Vancouver Canada). The ethanol in the liposome formulation was next removed by distillation under vacuum at 25 0 C.
  • EXAMPLE 3 Effect of Charge Ratio on Chemical and Physical Stability
  • the effect of the positive to negative charge ratio in liposomal PNT-100 formulations on the physical and chemical stability of the formulations was investigated.
  • the cationic cardiolipin analog (R)- PLC-2 has 2 positive charges
  • DOPC is a zwitterionic lipid with one positive and one negative charge
  • PNTlOO has 23 negative charges associated with the phosphate backbone and a number of positive charges associated with the bases that varies with the pH.
  • the charge ratios were varied from 1.5 to 2.5. (1.5, 2.0, 2.5) by varying the amount of (R)-PLC-2, DOPC and cholesterol while keeping the total lipids at 30.0 mg/ml and maintaining constant molar composition of 30.0% cholesterol.
  • the concentration of PNTlOO, (+)- ⁇ -tocopherol acid succinate and sucrose were kept constant.
  • the physical and chemical stabilities of the formulations were monitored at storage conditions of 2-8 0 C for up to 9 weeks. The results showed that a charge ratio of 2.0 is necessary to maintain the physical stability of the formulation. At a lower charge ratio the mean liposome diameter and 99 percentile of the size distribution increased with the storage time. Both 2.0 and 2.5 charge ratios provided stable formulations.
  • a charge ratio of 2.0 is preferred due to possible toxicity usually associated with increased amount of cationic lipids (or high positive charge) in liposomal formulations.
  • EXAMPLE 4 Effect of pH on Chemical and Physical Stability
  • Toxicity of a liposomal PNT-100 (SEQ ID NO:1251) formulation was performed in outbred cd-1 (normal) mice at multiple dose levels. Doses of 3.125, 6.25, 12.50 and 25.0 mg/kg of body weight were administered intravenously via the tail vein daily for 5 consecutive days. A 5% dextrose solution served as a control. Body weights, mortality and clinical signs were monitored to evaluate the severity of toxicity of liposomal PNT-100 for 22 days. No clinical signs of toxicity were observed in the surviving animals that were dosed with 5% dextrose or 3.125, 6.25 and 12.5 mg/kg of liposomal PNT-100.
  • the liposomal PNT-100 formulation termed PNT2252, was tested for anti-PC-3 efficacy in BALB/c nude mice.
  • PC-3 cells are a human prostate carcinoma cell line (ATCC CRL 1435).
  • Xenografts were generated from sub cutaneous injection of 2 X 10 6 PC-3 cells into BALB/c nude mice.
  • a liposomal PNT-100 mixture was formulated with a cardiolipin [(R)-PLC-2], DOPC and cholesterol at a molar ratio of DOPC/(R)-PLC-2/cholesterol of about 54.0/16.0/30.0.
  • the total lipids, lipid to PNT-100 molar ratio and (+/-) charge ratio are about 30.0 mg/ml, 144 and 2.0, respectively. There was no detectable free oligonucleotide or lipid material in the liposomal-PNT-100 mixture. The average particle size is 150 ⁇ m. [0343] The mice were divided into 6 groups and treated as indicated in Table 3. Tumor size was measured by caliper monitoring. Mice bearing 50-100 mm 3 xenografts received the treatment indicated in Table 3. The growth rate of the tumors was measured for 22 days post injection.
  • a single lOmg/kg LV. bolus dose of PNT2252 (Group 4) resulted in 8/15 animal deaths between eight and twenty hours post drug treatment. The surviving seven animals were given a second 7.5 mg/kg injection. The toxicity was hypothesized to result from PNT2252-blood aggregation. The difference observed between the toxic dose levels between outbred CD-I mice (See example 6) and the nude mice was attributed to the immunocompromised background of the nude animals.
  • PNT2252 aggregation effect was both toxic and inhibitory for bolus delivered PNT2252 efficacy. It is notable that the expected apoptotic-like tumor response to intratumor injected PNT2252 is significant. The toxicity is likely due to the cardiolipin formulation because a study has been completed that predictably demonstrated no anti-PC-3 xenograft activity and no toxicity from a single 100 mg/kg bolus dose of naked PNTlOO oligonucleotide.
  • a pilot PNT2252 toxicology study was performed that examined whether PNT2252 toxicity could be diminished if delivered by LV. one hour infusion. The study demonstrated that 20 mg/kg/day qdX5 of PNT2252 could be administered by infusion delivery to normal BALB/c mice without a demonstrable toxic response.
  • WSU-DLCL 2 Wayne State University diffuse large cell lymphoma
  • WSU-DLCL 2 cells were obtained from Dr. Ramzi Mohammad and Dr. Al-Katib and colleagues at the Karmanos Cancer Institute at Wayne State University. (See Al-Katib, AM, et al., Clin. Cancer Res. 4, 1305-1314 (1998); Mohammad, R, et al., Clin. Cancer Res. 8, 1277-1283 (2002); Mohammad, RM, et al., MoI. Cancer Ther., 4, 13-21 (2005); Mohammad, RM, et al., Clin. Cancer Res. 6, 4950-4956, (2000).)
  • Results are shown in Figure 2 and indicate that the growth rate of the cells that received PNTlOO data is 56% of the growth rate of cells treated with the PNTl control (SEQ ID NO: 1448) and 51% of the growth rate of cells receiving no treatment.
  • PNT104 is SEQ ID NO: 1250.
  • PC-3 cells are a widely used model of hormone refractory prostate cancer. The effect of liposomal PNTlOO on the proliferation of PC-3 cells was determined. Low passage PC-3 cells were obtained and cultured according to ATCC recommendations. Cells were seeded in 6-well plates for 48 hrs in complete serum containing media, incubated for two hours in fresh media, and the media was replaced with serum free media.
  • NeoPhectin was combined according to manufacturer's instructions (NeoPharm) with the indicated concentrations of PNTlOO (SEQ ID NO:1251), PNT104 (methylated CpG PNTlOO, SEQ ID NO:1250), PNTl (24mer scrambled oligonucelotide control (SEQ ID NO: 1448), PNT102 (24mer mismatch control with a nucleotide 21 G to A mutation, SEQ ID NO: 1449), and PNTlOOR (24mer PNTlOO reverse complement control, SEQ ID NO: 1288). The ratio was 6:1 DNA:NeoPhectin.
  • oligonucleotides 10 ⁇ M final concentration of oligonucleotides was incubated with cells for 6 hrs at 37 0 C. Cells were then returned to complete media and fed daily for 48 hours. After the indicated incubation time, the CellTiter-GloTM luminescent cell viability assay was performed according to manufacture's instructions (Promega, Madison, WI). This assay uses a luciferase reaction to measure ATP, a global indicator of cellular metabolism. Statistical cell viability was calculated using a student-t test with 95% confidence with two-way ANOVA analysis and Bonferroni paired comparisons.
  • Xenografts were generated in CB- 17 SCID mice as described in example 7, except that WSU-
  • NeoPhectin-AT A 25 ml liposome delivery vehicle (LDV) consisting of NeoPhectin-AT (NeoPharm, IL) bottle was placed at room temperature for 15 min. The bottle was gently swirled for 30 seconds to mix. 1000 ⁇ l LDV was transferred to 50 ml sterile polypropylene tubes labeled: Day # PNTlOO. The PNTlOO stock tube was vortexed and quickly centrifuged.
  • LDV liposome delivery vehicle
  • BCL2 Western blot analysis was performed with xenografts harvested from additional animals 72 hours post drug treatment to determine a targeted drug response. Total protein extracts were prepared from two xenografts from each treatment group. The protein extracts were pooled and equal amounts were used for Western blot analysis with anti-BCL2 and anti- GAPDH (G3PDH; loading control) antibodies. Results in figure 4B show apparent diminished BCL2 levels compared to controls. BCL2 levels match the xenograft response described in Figure 4A.
  • EXAMPLE 11 Efficacy of Liposomal PNT- 100 and TaxotereTM in a PC-3 Xenograft Model [0360] The effect of PNT-100 formulated in NeoPhectin-AT was determined in PC-3 xenografts. PC-3 xenografts were generated by subcutaneous injection of 2x10 6 PC-3 cells in nude mice. A 6:1 PNT100:NeoPhectin AT charge ratio was prepared as described in Example 10.
  • mice bearing 50-100 mm 3 xenografts were dosed intravenously with 1 mg/kg PNT-100 + NeoPhectin AT, daily for 5 days, with 10 mg/kg on day 2 and with 5 m/kg on day 5 with TaxotereTM. Tumor response was measured by caliper monitoring. Results are shown in Figure 5, which indicate PNT-100 slows that PNT-100 with TaxotereTM is more efficacious than PNT-100 or TaxotereTM alone.
  • EXAMPLE 12 Effect of c-mvc Oligonucleotides on the Growth of Cultured cells
  • Human breast cancer cells MCF-7, MDA-MB-231 and MCF-12A were obtained from the Karmanos Cancer Institute. The cells were cultured in DMDM/F12 media supplemented with 10 mM HEPES and 29mM sodium bicarbonate.
  • CM4 SEQ ID NO.940, CM7 (SEQ ID NO:943) and PNT-I (SEQ ID NO: 1448) were prepared with NeoPhectin as described in Example 8. Cells were grown, treated with the oligonucleotides and the growth of the cells measured as described in Example 8.
  • CM-7 SEQ ID NO:943
  • NeoPhectin slows the growth of MCF-7 cells compared to controls, including a scrambled oligonucleotide formulated with NeoPhectin.

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Abstract

Cette invention concerne des compositions et des méthodes visant à inhiber l'expression génique. Cette invention concerne en particulier des oligonucléotides séquestrés dans des liposomes cationiques servant au traitement du cancer.
PCT/US2006/046298 2005-12-01 2006-12-01 Systeme d'administration de liposomes cationiques a oligonucleotides Ceased WO2007065017A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8367628B2 (en) 2005-12-01 2013-02-05 Pronai Therapeutics, Inc. Amphoteric liposome formulation
US8815599B2 (en) 2004-06-01 2014-08-26 Pronai Therapeutics, Inc. Methods and compositions for the inhibition of gene expression
WO2021055660A1 (fr) * 2019-09-18 2021-03-25 The Regents Of The University Of California Nanoliposomes produisant des protéines et leurs utilisations
US12004868B2 (en) 2011-06-03 2024-06-11 Signpath Pharma Inc. Liposomal mitigation of drug-induced inhibition of the cardiac IKr channel

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US6977244B2 (en) * 1996-10-04 2005-12-20 Board Of Regents, The University Of Texas Systems Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
US20030087861A1 (en) * 2001-05-17 2003-05-08 Iversen Patrick L Combined approach to treatment of cancer using a c-myc antisense oligomer
EP1556392A1 (fr) * 2002-10-16 2005-07-27 Neopharm, Inc. Molecules de cardiolipine et procedes pour leur synthese
AU2005204689A1 (en) * 2004-01-07 2005-07-28 Neopharm, Inc. Lipid compositions and use thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8815599B2 (en) 2004-06-01 2014-08-26 Pronai Therapeutics, Inc. Methods and compositions for the inhibition of gene expression
US9393258B2 (en) 2004-06-01 2016-07-19 Pronai Therapeutics, Inc. Methods and compositions for the inhibition of gene expression
US8367628B2 (en) 2005-12-01 2013-02-05 Pronai Therapeutics, Inc. Amphoteric liposome formulation
US12004868B2 (en) 2011-06-03 2024-06-11 Signpath Pharma Inc. Liposomal mitigation of drug-induced inhibition of the cardiac IKr channel
WO2021055660A1 (fr) * 2019-09-18 2021-03-25 The Regents Of The University Of California Nanoliposomes produisant des protéines et leurs utilisations

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